Non-Canonical NF-Kb Signaling Regulates Hematopoietic Stem Cell Self-Renewal and Microenvironment Interactions

Abstract Abstract 859 RelB and NF-kB2 are the main effectors of NF-kB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-kB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-kB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

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U2AF1(S34F) Mutant Hematopoietic Cells Require Expression of Wild-Type U2af1 for Survival

Blood ◽

10.1182/blood-2018-99-110836 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 941-941

Author(s):

Brian Wadugu ◽

Amanda Heard ◽

Joseph Bradley ◽

Matthew Ndonwi ◽

Jin J Shao ◽

...

Keyword(s):

Bone Marrow ◽

Rna Splicing ◽

Bone Marrow Cells ◽

Hematopoietic Cells ◽

Poly I:C ◽

Wild Type ◽

Hematopoietic Stem ◽

Mutant Cells ◽

Marrow Cells

Abstract Somatic mutations in U2AF1, a spliceosome gene involved in pre-mRNA splicing, occur in up to 11% of MDS patients. While we reported that mice expressing mutant U2AF1(S34F) have altered hematopoiesis and RNA splicing, similar to mutant MDS patients, the role of wild-type U2AF1 in normal hematopoiesis has not been studied. U2AF1mutations are always heterozygous and the wild-type allele is expressed, suggesting that mutant cells require the residual wild-type (WT) allele for survival. A complete understanding of the role of wild-type U2AF1 on hematopoiesis and RNA splicing will enhance our understanding of how mutant U2AF1 contributes to abnormal hematopoiesis and splicing in MDS. In order to understand the role of wild-type U2af1 in normal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse (U2af1flox/flox). Homozygous embryonic deletion of U2af1using Vav1-Cre was embryonic lethal and led to reduction in fetal liver hematopoietic stem and progenitor cells (KLS and KLS-SLAM, p ≤ 0.05) at embryonic day 15, suggesting that U2af1 is essential for hematopoiesis during embryonic development. To study the hematopoietic cell-intrinsic effects of U2af1 deletion in adult mice, we performed a non-competitive bone marrow transplant of bone marrow cells from Mx1-Cre/U2af1flox/flox, Mx1-Cre/U2af1flox/wtor Mx1-Cre/U2af1wt/wtmice into lethally irradiated congenic recipient mice. Following poly I:C-induced U2af1deletion, homozygous U2af1 KOmice, but not other genotypes (including heterozygous KO mice), became moribund. Analysis of peripheral blood up to 11 days post poly I:C treatment revealed anemia (hemoglobin decrease >1.7 fold) and multilineage cytopenias in homozygous U2af1 KOmice compared to all other genotypes(p ≤ 0.001, n=5 each).Deletion of U2af1 alsoled to rapid bone marrow failure and a reduction in the absolute number of bone marrow neutrophils (p ≤ 0.001), monocytes (p ≤ 0.001), and B-cells (p ≤ 0.05), as well as a depletion of hematopoietic progenitor cells (KL, and KLS cells, p ≤ 0.001, n=5 each). Next, we created mixed bone marrow chimeras (i.e., we mixed equal numbers of homozygous KO and wild-type congenic competitor bone marrow cells and transplanted them into lethally irradiated congenic recipient mice) to study the effects of U2af1 deletion on hematopoietic stem cell (HSC) function. As early as 10 days following Mx1-Cre-induction, we observed a complete loss of peripheral blood neutrophil and monocyte chimerism of the U2af1 KOcells, but not U2af1 heterozygous KO cells, and at 10 months there was a complete loss of homozygous U2af1 KObone marrow hematopoietic stem cells (SLAM, ST-HSCs, and LT-HSCs), neutrophils, and monocytes, as well as a severe reduction in B-cells and T-cells (p ≤ 0.001, n=3-4 for HSCs. p ≤ 0.001, n=9-10 for all other comparisons). The data indicate that normal hematopoiesis is dependent on wild-type U2af1expression, and that U2af1 heterozygous KO cells that retain one U2af1 allele are normal. Next, we tested whether mutant U2AF1(S34F) hematopoietic cells require expression of wild-type U2AF1 for survival. To test this, we used doxycycline-inducible U2AF1(S34F) or U2AF1(WT) transgenic mice. We generated ERT2-Cre/U2af1flox/flox/TgU2AF1-S34F/rtTA(S34F/KO), and ERT2-Cre/U2af1flox/flox/TgU2AF1-WT/rtTA,(WT/KO) mice, as well as all other single genotype control mice. We then created 1:1 mixed bone marrow chimeras with S34F/KO or WT/KO test bone marrow cells and wild-type competitor congenic bone marrow cells and transplanted them into lethally irradiated congenic recipient mice. Following stable engraftment, we induced U2AF1(S34F) (or WT) transgene expression with doxycycline followed by deletion of endogenous mouse U2af1 using tamoxifen. As early as 2 weeks post-deletion of U2af1, S34F/KO neutrophil chimerism dropped to 5.4% indicating loss of mutant cells, while WT/KO neutrophil chimerism remained elevated at 31.6% (p = 0.01, n=6-8). The data suggest that mutant U2AF1(S34F) hematopoietic cells are dependent on expression of wild-type U2af1 for survival. Since U2AF1mutant cells are vulnerable to loss of the residual wild-type U2AF1allele, and heterozygous U2af1KO cells are viable, selectively targeting the wild-type U2AF1allele in heterozygous mutant cells could be a novel therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

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Protective Role of Connexin 32 in Experimental Leukemogenesis.

Blood ◽

10.1182/blood.v110.11.2239.2239 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2239-2239

Author(s):

Yoko Hirabayashi ◽

Byung-Il Yoon ◽

Isao Tsuboi ◽

Yan Huo ◽

Yukio Kodama ◽

...

Keyword(s):

Bone Marrow ◽

Steady State ◽

Bone Marrow Cells ◽

Pcr Analysis ◽

Wild Type ◽

Hematopoietic Stem ◽

Immunomagnetic Bead ◽

Competitive Assay ◽

Marrow Cells

Abstract Connexin (Cx) functions in the organization of cell-cell communication in multicellular organisms. Gap junctions have been implicated in the homeostatic regulation of various cellular functions, including growth control and differentiation, apoptosis, and the synchronization of electrotonic and metabolic functions. Primitive hemopoietic progenitor cells form a multicellular system, but a previous report describes that Cx32 is not expressed in the bone marrow. Thus, a question arises as to why Cx molecules are not detected in the hematopoietic tissue other than stromal cells. Based on our preliminary study that suggested a potential impairment of hematopoiesis in Cx32-knockout (KO) mice, the objectives of the present study were to determine whether Cx32 functions in the bone marrow during steady-state hematopoiesis and further to examine its possible protective roles during regeneration after chemical abrasions and during leukemogenesis after the administration of a genotoxic chemical, methyl nitrosourea (MNU). As results, the Cx32 molecule functioning in the hematopoietic stem cell (HSC) compartment during steady-state hematopoiesis was observed for the first time; the expression of Cx32 at the mRNA level determined by PCR analysis and that at the protein level determined using an anti-Cx32 antibody were observed only in the lin−c-kit+ HSC fraction using a combination of immunobead-density gradient and immunomagnetic-bead separation. Hematopoiesis was impaired in the absence of Cx32; it was delayed during regeneration after chemical abrasion with 5-fluorouracil at 150 mg/kg body weight in Cx32-KO mice. Cx32-KO mice also showed increased leukemogenicity compared with wild-type mice after MNU injection; furthermore, in a competitive assay for leukemogenicity in mice that had been lethally irradiated and repopulated with a mixed population of equal amount of bone marrow cells from Cx32-KO mice and wild-type mice, the resulting leukemias were originated predominantly from Cx32-KO bone marrow cells. The present competitive assay clearly showed that Cx32-KO bone marrow cells have a higher risk of becoming leukemogenic. The above-mentioned findings in this study imply that Cxs play an essential role in maintaining the steady-state hematopoiesis and suppressing the neoplastic change. In summary, the role of Cx32 in hematopoiesis was not previously recognized and Cx32 was expressed only in HSCs and their progenitors. The results indicate that Cx32 in wild-type mice protects HSCs from chemical abrasion and leukemogenic impacts. Our results indicate that the risk of developing leukemia in patients with X-chromosome-linked Cx32 deficiency, called Charcot-Marie-Tooth syndrome, may not be incidental.

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R-Ras Regulates Hematopoietic Stem/Progenitor Cell Adhesion, Migration, and Mobilization through Rac GTPase-Mediated Signals.

Blood ◽

10.1182/blood.v110.11.221.221 ◽

2007 ◽

Vol 110 (11) ◽

pp. 221-221

Author(s):

Xun Shang ◽

Lina Li ◽

Jose Concelas ◽

Fukun Guo ◽

Deidre Daria ◽

...

Keyword(s):

Bone Marrow ◽

Cell Adhesion ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Wild Type ◽

Rac Gtpase ◽

Hematopoietic Stem ◽

And Migration ◽

Marrow Cells

Abstract Hematopoietic stem/progenitor cells (HSPCs) are maintained by strictly regulated signals in the bone marrow microenvironment. One challenge in understanding the complex mode of HSPC regulation is to link intracellular signal components with extracellular stimuli. R-Ras is a member of the Ras family small GTPases. Previous mouse genetic studies suggest that R-Ras mRNA is primarily expressed in endothelial cells and R-Ras is involved in vascular angiogenesis. In clonal cell lines, although dominant mutant overexpression studies suggest a possible role of R-Ras in regulating cell adhesion and spreading, proliferation and/or differentiation in a cell-type dependent manner, it remains controversial whether R-Ras activity may promote or inhibit cell adhesion and migration. Here, in a mouse knockout model, we have examined the role of R-Ras in HSPC regulation by a combined in vivo and in vitro approach. Firstly, we found that R-Ras is expressed in the Lin− low density bone marrow cells of wild-type mice, and R-Ras activity in the cells is downregulated by cytokines and chemokines such as SCF and SDF-1a (∼ 20% and 40% of unstimulated control, respectively). Secondly, R-Ras deficiency did not significantly affect peripheral blood CBC, nor alter the frequency or distribution of long-term and short-term hematopoietic stem cells (defined by IL7Ra−Lin−Sca-1+c-Kit+CD34− and IL7Ra−Lin−Sca-1+c-Kit+CD34+ genotypes, respectively) in the bone marrow, peripheral blood and spleen. Competitive repopulation experiments using the wild-type and R-Ras−/− bone marrow cells at 1:1 ratio in lethally irradiated recipient mice showed no significant difference of blood cells of the two genotypes in the recipients up to 6 months post-transplantation. R-Ras−/− bone marrow cells did not show a detectable difference in colony forming unit activities assayed in the presence of various combinations of SCF, TPO, EPO, IL3, G-CSF and serum, compared with the matching wild-type cells. Thirdly, upon challenge with G-CSF, a HSPC mobilizing agent, R-Ras−/− mice demonstrated a markedly enhanced ability to mobilize HSPCs from bone marrow to peripheral blood as revealed by genotypic and colony-forming unit analyses (WT: 150 vs. KO: 320 per 200uL blood, p=0.018), and R-Ras−/− HSPCs exhibit significantly decreased homing activity (WT: 4.3% vs. KO: 2.8%, p<0.001). Fourthly, isolated R-Ras−/− HSPCs displayed a constitutively assembled cortical actin cytoskeleton structure in the absence of cytokine or chemokine stimulation, similar to that of activated wild-type HSPCs. The R-Ras−/− HSPCs were defective in adhesion of cobblestone area-forming cells to a bone marrow-derived stroma cell line (FBMD-1) and in adhesion to fibronectin CH296 fragment, and showed a drastically increased ability to migrate toward a SDF-1a gradient (WT: 16% vs. KO: 38%, p<0.001). These data point to a HSPC-intrinsic role of R-Ras in adhesion and migration. Finally, the functional changes of R-Ras−/− cells were associated with a ∼3 fold increase in Rac-GTP species and constitutively elevated Rac downstream signals of phsopho-PAK1 and phospho-myosin light chain. Partial inhibition of Rac activity by NSC23766, a Rac GTPase-specific inhibitor, readily reversed the migration phenotype under SDF-1a stimulation. Taken together, these studies demonstrate that R-Ras is a critical signal regulator for HSPC adhesion, homing, migration, and mobilization through a mechanism involving Rac GTPase-regulated cytoskeleton and adhesion machinery.

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Mutant p53 Drives the Development of Pre-Leukemic Hematopoietic Stem Cells through Modulating Epigenetic Regulators

Blood ◽

10.1182/blood.v126.23.307.307 ◽

2015 ◽

Vol 126 (23) ◽

pp. 307-307

Author(s):

Sarah C Nabinger ◽

Michihiro Kobayashi ◽

Rui Gao ◽

Sisi Chen ◽

Chonghua Yao ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Leukemia Stem Cells ◽

Mutant P53 ◽

Self Renewal ◽

Hematopoietic Stem ◽

Tp53 Mutations ◽

Marrow Cells

Abstract AML is thought to arise from leukemia stem cells (LSCs); however, recent evidence suggests that the transforming events may initially give rise to pre-leukemic hematopoietic stem cells (pre-leukemic HSCs), preceding the formation of fully transformed LSCs. Pre-leukemic HSCs have been shown to contribute to normal blood development and harbor a selective growth advantage compared to normal HSCs. Pre-leukemic HSCs can acquire subsequent mutations, and once differentiation capacity is impaired, leukemia emerges. Recently, acquired somatic TP53 mutations, including p53R248W and p53R273H, were identified in healthy individuals as well as AML patients, suggesting that TP53 mutations may be early events in the pathogenesis of AML. We found that p53R248W HSCs showed a multi-lineage repopulation advantage over WT HSCs in transplantation experiments, demonstrating that mutant p53 confers a pre-leukemic phenotype in murine HSCs. Although TP53 mutations are limited in AML, TP53 mutations do co-exist with mutations of epigenetic regulator, ASXL-1, or receptor tyrosine kinase, FLT3, in AML. Mutations in Asxl-1 are present in ~10-30% of patients with myeloid malignancies and confer poor prognosis. Loss of Asxl-1 in the hematopoietic compartment leads to a myelodysplastic-like syndrome in mice and reduced stem cell self-renewal. Internal tandem duplications in Flt3 (Flt3-ITD) occur in ~30% of AML patients and are associated with adverse clinical outcome. Flt3-ITD-positive mice develop a myeloproliferative neoplasm (MPN) and HSCs expressing Flt3-ITD have decreased self-renewal capabilities. We hypothesize that mutant p53 drives the development of pre-leukemic HSCs with enhanced self-renewal capability, allowing clonal expansion and subsequent acquisition of Asxl-1 or Flt3 mutations leading to the formation of fully transformed leukemia stem cells. To define the role of mutant p53 in Asxl-1+/- HSCs, we generated p53R248W/+ Asxl-1+/- mice and performed in vitro serial replating assays as well as in vivo competitivebone marrow transplantation experiments. We found that p53R248W significantly enhanced the serial replating ability of Asxl-1-deficient bone marrow cells. Interestingly, while bone marrow from Asxl-1+/- mice had very poor engraftment compared to wild type bone marrow cells 16 weeks post-transplantation, the expression of p53R248W in Asxl-1+/- bone marrow rescued the defect. To examine the role of mutant p53 in Flt3-ITD-positive HSCs, we generated p53R248W/+ Flt3ITD/+ mice. We found that p53R248W enhanced the replating ability of Flt3ITD/+ bone marrow cells. Despite the fact that Flt3ITD/+ bone marrow cells displayed decreased repopulating ability compared to wild type cells 16 weeks post-transplant, expression of p53R248W in Flt3ITD/+ cells rescued the defect. We are monitoring leukemia development in primary and secondary transplant recipients as well as in de novo p53R248W/+ Asxl-1+/- and p53R248W/+ Flt3ITD/+ animals and predict that mutant p53 may cooperate with Asxl-1 deficiency or Flt3-ITD in the formation of LSCs to accelerate leukemia development in Asxl-1 deficient or Flt-ITD-positive neoplasms. Mechanistically, dysregulated epigenetic control underlies the pathogenesis of AML and we discovered that mutant p53 regulates epigenetic regulators, including Ezh1, Ezh2, Kdm2a, and Setd2, in HSCs. H3K27me3 is catalyzed by EZH1 or EZH2 of the Polycomb repressing complex 2 (PRC2). Both Ezh1 and Ezh2 are important for HSC self-renewal. SETD2 is a histone H3K36 methyltransferase and mutations in SETD2 have been identified in 6% of patients with AML. SETD2 deficiency resulted in a global loss of H3K36me3 and increased self-renewal capability of leukemia stem cells. We found that there were increased levels of H3K27me3 and decreased levels of H3K36me3 in p53R248W/+ HSCs compared to that of the WT HSCs. In ChIP experiments, we found that p53R248W, but not WT p53, was associated with the promoter region of Ezh2 in mouse myeloid progenitor cells, suggesting that p53R248W may directly activate Ezh2 expression in hematopoietic cells. Given that Asxl-1 has been shown to regulate H3K27me3 in HSCs, the synergy between mutant p53 and Asxl-1 deficiency on LSC self-renewal could be due to changes in histone modifications. Overall, we demonstrate that mutant p53 promotes the development of pre-leukemic HSCs by a novel mechanism involving dysregulation of the epigenetic pathways. Disclosures No relevant conflicts of interest to declare.

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Effects of the Homeodomain Gene Pitx2 on Self-Renewal of Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v110.11.95.95 ◽

2007 ◽

Vol 110 (11) ◽

pp. 95-95 ◽

Cited By ~ 1

Author(s):

Hui Z. Zhang ◽

Svetlana Rogulina ◽

Wendy Chen ◽

Barbara A. Degar ◽

Bernard G. Forget

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Leukemic Cells ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic Stem ◽

Colony Forming Units ◽

Marrow Cells

Abstract Pitx2, a homeodomain gene preferentially expressed in murine hematopoietic stem/progenitor cells, is also a downstream target of genes important for hematopoiesis such as MLL and Wnt/Dvl/β-Catenin. We have previously reported that Pitx2 null hematopoietic stem cells (HSCs) can contribute to multi-lineage hematopoiesis under physiologic conditions. We have now carried out serial bone marrow transplantation experiments and demonstrated that after the 3rd round of serial transplantation, Pitx2 null cells reconstituted only 28.6% of the recipient hematopoietic cells as compared to 60% in the case of wild type cells (P<0.001). There were no Pitx2 null donor-derived cells in recipient mice after the 4th round of transplantation, whereas donor-derived chimerism was 57% with wild type cells (P<0.001), and 26% with Pitx2 +/− cells (P<0.001). Therefore, Pitx2 null HSCs have decreased self renewal capacity. To further study the function of Pitx2 in HSC, we constitutively overexpressed the Pitx2 gene in murine bone marrow cells following transduction using a MSCV/IRES/GFP retroviral vector, and analyzed the effects on hematopoiesis in vitro and in vivo. Bone marrow cells overexpressing Pitx2 were isolated on the basis of their GFP expression and analyzed for their colony forming ability in vitro. Retrovirally transduced bone marrow cells were also transplanted into lethally irradiated mice, and the transplanted mice were observed for long-term reconstitution. Colony-forming unit assays showed that Pitx2 overexpressing bone marrow cells, compared to control cells transduced with vector only, had increased numbers of GM colony forming units and reduced numbers of megakaryocytic colony forming units. Pitx2-overexpressing cells continued to form GM colonies after more than eight serial replatings. When these cells were cultured in liquid medium containing SCF, IL-3 and IL-6, they gave rise to cells that stained positively either for alpha naphthyl butyrate, indicating monocytic differentiation, or for peroxidase, indicating neutrophilic differentiation. The ability of these GM-colony forming cells to cause leukemia is currently under investigation. Long-term reconstitution of hematopoiesis in mice by Pitx2 over-expressing HSCs was demonstrated by identifying GFP positive multi-lineage peripheral blood cells four months following transplantation. One of these mice manifested leukemia at this time, as evidenced by a markedly elevated WBC count and other hematologic abnormalities. The leukemic WBCs had very high levels of GFP and Pitx2 expression and were shown to contain two retroviral integration sites, neither of which involved a known oncogene or overexpression of the gene at the integration site. Immunophenotyping by flow cytometry demonstrated that the majority of the leukemic cells were c-kit positive and expressed the megakaryocytic marker CD41, as well as the common myeloid progenitor marker, CD16/32. Some of the cells expressed the erythroid marker Ter119. The leukemic cells did not express any lymphoid markers, including CD3ε, B220, CD19, and IL7R3. This Pitx2-overexpression-associated leukemia was transplantable. Experiments are under way to characterize the leukemia initiating cells. Taken together, our results provide evidence that the homeodomain gene Pitx2 plays a role in the self-renewal of hematopoietic stem/progenitor cells.

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MEIS1 Is Required for the Maintenance of Long Term Hematopoietic Stem Cells Wherein It Regulates Quiescence

Blood ◽

10.1182/blood.v118.21.551.551 ◽

2011 ◽

Vol 118 (21) ◽

pp. 551-551

Author(s):

Zeenath Unnisa ◽

Jason P Clark ◽

Elizabeth Wojtowicz ◽

Lino Tessarollo ◽

Neal G. Copeland ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Bone Marrow Analysis ◽

Marrow Cells

Abstract Abstract 551 Normal hematopoiesis is maintained by long-term hematopoietic stem cells (LT-HSCs) that are defined by their extensive self-renewal and multipotency. Self-renewal of LT-HSCs in turn is regulated by a complex network of intrinsic and extrinsic factors. The transcription factor MEIS1 is highly expressed in hematopoietic stem and progenitor cells and also in several leukemias, suggesting that MEIS1 might be important in regulating self-renewal. However, the role of MEIS1 in normal hematopoiesis has not been defined. To determine the role of MEIS1 in hematopoiesis, we studied conditional knockout mice. We generated transgenic mice bearing loxp sites flanking the homeodomain of MEIS1. The MEIS1-floxed mice were then bred to Rosa26-CreERT2 mice, the latter expressing cre-recombinase ubiquitously, that can be activated by estrogen or its analog Tamoxifen (Tam). Efficient, complete recombination was achieved in vivo by treating MEIS1-f/f-Cre (homozygous for MEIS1-flox) mice with Tam and in vitro by treating bone marrow cells with 4-hydroxy tamoxifen. Loss of MEIS1 expression was detected by QRT-PCR and western blotting. To determine the role of MEIS1 in the maintenance of adult hematopoiesis, MEIS1-f/f-Cre and control mice were treated with Tam and MEIS1 deletion confirmed by PCR. At three weeks post deletion, bone marrow analysis showed a significant reduction in the number of LT-HSCs defined as lin-/c-Kit+/Sca1+/CD48−/CD150+ in the MEIS1-depleted mice compared to controls (0.012% compared to 0.037%, N=6, p<0.05, t-test). However, the progenitor populations were unaffected by MEIS1 deletion. Over a period of 12 weeks of observation, the mice did not show any signs of distress and the peripheral blood counts of the experimental and control mice remained normal, indicating that short term hematopoiesis was not affected. Cell cycle analysis of LT-HSCs showed that MEIS1 deletion resulted in a significant shift of cells from G0 to G1 phase (G0 and G1 proportions respectively, 81.75±3.25% and 9.40±3% for control and 56.10±0.873% and 31.17±1.5% for MEIS1-deleted). To determine the effects of MEIS1 loss on intrinsic hematopoietic stem cell function, we performed competitive repopulation assays. Bone marrow cells harvested from MEIS1-f/f-Cre or MEIS1-f/+-Cre (control) mice were combined with equal numbers of bone marrow cells from BoyJ mice and transplanted via tail vein injection into lethally irradiated BoyJ mice. Four weeks after transplant, recipients were treated with Tam or vehicle for 5 days and deletion of MEIS1 confirmed by PCR on peripheral blood. Peripheral blood of recipient mice was analyzed at 1, 4, 8, 12 and 16 weeks after treatment and relative chimerism assessed by flow cytometry. At 1 and 4 weeks after treatment, the chimerism in the MEIS1 deleted group (Tam treated MEIS1-f/f-CreER) and the control groups (Tam treated MEIS1-f/+-CReER and vehicle treated MEIS1-f/f-CreER) was comparable (41%, 40.5% and 41.5% respectively, average, N=5 to 8). However, by 8 weeks after treatment, the MEIS1 deleted group showed a significant decline in chimerism compared to controls (18.2% compared to 43.1% and 35.1% respectively, p<0.02, t-test) and at 16 weeks the chimerism in the MEIS1-deleted group declined further (11.1% compared to 40.2% and 35.0% respectively, p<0.001). Subpopulation analysis showed loss of chimerism in granulocytes and in B and T lymphocytes. The latency and breadth of the effect of MEIS1 loss suggested an effect on the hematopoietic stem cell population. Indeed, bone marrow analysis of transplant recipients showed near complete loss of LT-HSC chimerism (3% compared to 70.25% and 75.6% respectively, p<0.001). Finally, we performed gene expression profiling on lineage negative bone marrow cells with and without MEIS1 deletion. Results showed that loss of MEIS1 was associated with decreased expression of hypoxia-responsive genes. Collectively, these results indicate that MEIS1 is required for the maintenance of the pool of LT-HSCs. Loss of MEIS1 promotes cycling and exhaustion of LT-HSCs. Further, we propose that activation of the hypoxia-response pathway may be one of the mechanisms by which MEIS1 exerts its effects on hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

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Loss of IKKβ Increases the Number and Self-Renewal Capacity of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v124.21.4345.4345 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4345-4345

Author(s):

Jing Zhang ◽

Li Li ◽

Albert S. Baldwin ◽

Alan D. Friedman ◽

Ido Paz-Priel

Keyword(s):

Bone Marrow ◽

Knockout Mice ◽

Bone Marrow Cells ◽

Fold Increase ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic Stem ◽

Pharmacologic Inhibition ◽

Wild Type Control ◽

Marrow Cells

Abstract NF-kB is an important regulator of both differentiation and function of lineage-committed hematopoietic cells. Targeted deletion of IkB kinase (IKK) β results in altered cytokine signaling and marked neutrophilia. To investigate the roles of IKKβ in regulation of hematopoiesis, we employed Mx1-Cre mediated IKKβ conditional knockout mice. As previously reported, deletion of IKKβ in hematopoietic cells results in neutrophilia, and decreased monocytes and. We now show that bone marrow cells from IKKβ deleted mice display accumulation of granulocyte-macrophage progenitors (GMPs, Lin-Sca-1-c-kit+CD34+CD16/32+), with 1.8-fold increase compared to wild-type control. Accordingly, methylcellulose-based colony-forming assay demonstrated that IKKβ-deficient marrow produced increased proportion of CFU-G and decreased CFU-M compared with wild type control. Importantly, pharmacologic inhibition of IKKβ activity in wild type murine bone marrow cells results in significant increase in the total number of colonies and the number and proportion of CFU-G colonies. In addition, loss of IKKβ is associated with a modest decrease of baseline hematocrit levels (52.5 ± 0.64 vs. 43.9 ± 4.3, p<0.0001) and blunted recovery after challenge with 5-FU. Accordingly, IKKβ deleted mice have 3-fold decreased megakaryocyte-erythrocyte progenitors population (MEPs, Lin-Sca-1-c-kit+CD34-CD16/32-). Using FACS analysis erythroid progenitor subsets in the marrow were characterized based on Ter119, CD71, and forward scatter. We found a significantly reduced frequency of proerythroblasts, basophilic and polychromatic erythroblasts. When cultured in methylcellulose in the presence of hEPO IKKβ-deficient bone marrow cells yielded a significantly decreased number of BFU-E compared to wild type. Accordingly, pharmacologic inhibition of IKKβ in wild type marrow cells resulted in diminished BFU-E colonies formation. We next studied the role of IKKβ in early hematopoietic progenitors. Bone marrow from IKKβ knockout mice displays the accumulation of phenotypic hematopoietic stem cells (HSCs), including LT-HSCs (Lin-Sca-1+c-kit+CD135-CD34-) and ST-HSCs (Lin-Sca-1+c-kit+CD135-CD34+), with 1.5-fold increase as compared to the wild-type control. Functionally, Lin- bone marrow cells from IKKβ deleted mice show increased serial replating in colony-forming assays, indicating increased cell autonomous long-term self-renewal capacity. Accordingly, competitive transplantation studies demonstrated that deletion of IKKβ greatly increases the repopulation ability of HSCs resulting in a stable advantage of bone marrow derived from IKKβ knockout mice. Quantitative real-time PCR assay demonstrated that compared to Lin- bone marrow from wild-type control mice, Lin- bone marrow cells from IKKβ deficient mice have up-regulation of genes related to HSC self-renewal in early stage of hematopoiesis and granulocytic lineage commitment such as GFI1, HOXA9, PU.1, C/EBPα, CEBPε; but down regulation of genes involved in megakaryocytic-erythroid lineage determination such as GATA1, GATA2, Tal-1, and Klf1. In summary, our data indicate that loss of IKKβ results in a cell autonomous alteration of expression of key regulators of hematopoiesis, leads to increased self-renewal of HSC, and drives hematopoietic development towards GMP lineage, favoring granulopoiesis over monopoiesis. Disclosures No relevant conflicts of interest to declare.

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Hmgb3 Deficiency Inhibits Down-Regulation of c-kit in Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v104.11.372.372 ◽

2004 ◽

Vol 104 (11) ◽

pp. 372-372

Author(s):

Michael J. Nemeth ◽

Stacie M. Anderson ◽

Lisa J. Garrett-Beal ◽

David M. Bodine

Keyword(s):

Bone Marrow ◽

Hind Limb ◽

Bone Marrow Cells ◽

Es Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Bone Marrow Cellularity ◽

Marrow Cellularity ◽

Marrow Cells

Abstract Hmgb3 is an X-linked member of a family of sequence-independent chromatin-binding proteins that is expressed in HSC-enriched lin−, c-kitHI, Sca-1HI, IL-7Rα− (KSIL) cells and Ter119+ erythroid cells. To define Hmgb3 function, we generated hemizygous mice (Hmgb3−/Y) using 129/SvJ ES cells. Hmgb3−/Y mice contain normal numbers of KSIL cells that are capable of normal repopulation and self-renewal. However, these mice have 1.6-fold fewer common lymphoid progenitors (CLP) and 3-fold fewer common myeloid progenitors (CMP) (p < 0.05). We hypothesized that the role of Hmgb3 in early hematopoiesis involves c-kit regulation. We observed that the level of c-kit mRNA in Hmgb3−/Y HSCs increased 30% compared to wild-type (WT) (p = 0.05). We used 5-fluorouracil (5-FU), which has been shown to down-regulate c-kit on HSCs, to characterize the interaction between Hmgb3 and c-kit. We monitored Hmgb3 expression in KSIL and lin−, Sca-1+, c-kit− cells before and after 5-FU treatment (150 mg/kg) using phenotypically normal transgenic mice containing an IRES-GFP cassette knocked into the 3′ UTR of Hmgb3. Prior to 5-FU treatment, 27% of KSIL cells were GFP+ (these cells were absent 4 days post-injection {p.i.}). In contrast, 1.8% of lin−, c-kit−, Sca-1+ cells were GFP+ before 5-FU treatment whereas 26% of lin−, c-kit−, Sca-1+ cells were GFP+ 4 days p.i. The increased proportion of GFP+ lin-, c-kit−, Sca-1+ cells after 5-FU treatment is consistent with previous findings that repopulating activity resides within the c-kit−/LO population in 5-FU treated bone marrow and our finding that Hmgb3 serves as a marker for long-term repopulating activity. To determine the time course of c-kit regulation, we compared bone marrow from 5-FU injected Hmgb3−/Y and WT mice for analysis at 2, 4, and 6 days p.i. Two days p.i., both WT and Hmgb3−/Y mice contained similar numbers of bone marrow cells (7 x 106 cells/hind limb) and the KSIL population was absent. By four days p.i., the bone marrow cellularity of WT mice declined to 5.5 ± 0.9 x 106 cells/hind limb and KSIL cells were still absent. However, in Hmgb3−/Y mice 4 days p.i., bone marrow cellularity stabilized at 7.9 ± 0.8 x 106 cells/hind limb, an increase of 43% compared to WT (p < 0.01), along with the re-emergence of the KSIL population. To determine whether the Hmgb3−/Y lin−, c-kit−, Sca-1+ population contains repopulating HSCs after 4 days of 5-FU treatment similar to WT mice, we performed repopulation assays using KSIL and lin−, c-kit−, Sca-1+ cells sorted from 4 day p.i. 5-FU treated Hmgb3−/Y mice. Recipients received either 2 x 104 KSIL or 2 x 105 lin−, c-kit−, Sca-1+ cells (Ly 5.2) from 5-FU treated Hmgb3−/Y mice along with a radioprotective dose of 3 x 105 congenic (Ly 5.1) bone marrow cells. FACS analysis performed on control recipients transplanted with congenic marrow exhibited < 1% Ly 5.2 cells in the bone marrow 16 weeks after transplant. Pre-5-FU treatment, 88% of bone marrow cells were donor derived in recipients of Hmgb3−/Y KSIL cells. There was no detectable engraftment of Hmgb3−Y lin−, c-kit−, Sca-1+ cells. In contrast to WT mice, both KSIL and lin−, c-kit−, Sca-1+ cells from 5-FU treated Hmgb3−/Y mice were capable of long-term repopulation (62–82% donor derived cells). We conclude that Hmgb3 deficiency facilitates the reemergence of c-kitHI HSCs following 5-FU treatment. Mechanisms involving either enhanced HSC self-renewal or delayed differentiation into CLPs and CMPs are both consistent with our results.

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DNMT3b’s Role in Hematopoietic Stem Cell Self-Renewal.

Blood ◽

10.1182/blood.v112.11.1406.1406 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1406-1406

Author(s):

Matthew J Boyer ◽

Feng Xu ◽

Hui Yu ◽

Tao Cheng

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Transplantation ◽

Peripheral Blood ◽

Marrow Transplantation ◽

Bone Marrow Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Marrow Cells

Abstract DNA methylation is an epigenetic means of gene regulation and is carried out by a family of methyltransferases of which DNMT1 acts to maintain methylation marks following DNA replication and DNMT3a and DNMT3b methylate DNA de novo. DNMT3b has been shown to be essential for mammalian development and necessary for differentiation of germline and neural progenitor cells. Mutations of DNMT3b in humans lead to a rare autosomal recessive disorder characterized by immunodeficiency, centromeric instability, and facial abnormalities. We have shown by real-time, RT-PCR that DNMT3b mRNA is uniquely over-expressed by approximately 30-fold in immunophenotypically-defined longterm repopulating hematopoietic stem cells (HSCs) that are CD34−lineage−c-kit+Sca-1+ as compared to progenitor and differentiated cell types within the bone marrow and with respect to the other members of the DNMT family, namely DNMT1 and DNMT3a. To determine DNMT3b’s function in HSCs competitive bone marrow transplantation was undertaken. Isolated lineage− enriched bone marrow cells were transduced with a retroviral backbone based on the Murine Stem Cell Virus (MSCV) carrying either GFP and a short, hairpin RNA (shRNA) targeting DNMT3b or GFP alone. Following transduction 1×105 GFP+ cells along with 1×105 competitor cells were transplanted into 9.5 Gray irradiated congenic recipients. Two months following transplantation mice receiving bone marrow cells transduced with DNMT3b shRNA showed a significantly lower engraftment of donor cells as a percentage of total competitor cell engraftment in the peripheral blood as compared to those receiving cells transduced with GFP alone (24.8 vs 3.7, p<0.05) which persisted at 3 months (22.8 vs 1.5, p<0.05). Similarly, within the donor derviced cells in the peripheral blood there was a lower percentage of myeloid (CD11b+) cells at 2 and 3 months in the recipients of DNMT3b shRNA transduced cells as compared to controls. However there was no observed difference in the percentage of peripheral B (CD45R+) or T (CD3+) cells within the donor-derived cells. To determine the mechanism behind the observed engraftment defect with DNMT3b knockdown we cultured GFP+ transduced bone marrow cells in vitro with minimal cytokine support. As a control for our targeting methodology we also transduced bone marrow cells from mice harboring two floxed DNMT3b alleles with a MSCV carrying Cre recombinase and GFP. While lineage− bone marrow cells transduced with GFP alone increased 10-fold in number over two weeks of culture, cells in which DNMT3b was down regulated by shRNA or Cre-mediated recombination only doubled. Culture of lineage− bone marrow cells in methylcellulose medium by the colony-forming cell (CFC) assay revealed increases in the granulocytic and total number of colonies with DNMT3b knockdown or Cre-mediated recombination of DNMT3b similar to the increased myeloid engraftment of DNMT3b shRNA transduced cells observed 1 month following competitive bone marrow transplantation. However when 5,000 of these cells from the first CFC assay were sub-cultured there was a significant loss of colony forming ability within all lineages when DNMT3b was targeted by shRNA or Cre-mediated recombination. Taken together with the decreased engraftment of DNMT3b shRNA cells following competitive bone marrow transplantation, the observed limited proliferation in liquid culture and loss of colony forming ability during serial CFC assays is suggestive of a self-renewal defect of HSCs in the absence of DNMT3b, that was previously only reported in the absence of both DNMT3a and DNMT3b. Further elucidation of this proposed self-renewal defect is being undertaken and results of ongoing studies including long-term culture initiating cell (LTC-IC) assays and identification of genomic sites of DNA methylation within different hematopoietic subsets will also be presented.

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Understanding the Impact of Inflammation on Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v116.21.2629.2629 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2629-2629

Author(s):

Ying Zhao ◽

Flora Ling ◽

Hong-Cheng Wang ◽

Xiao-Hong Sun

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Chronic Inflammation ◽

Bone Marrow Cells ◽

Wild Type ◽

Hematopoietic Stem ◽

Inflammatory Conditions ◽

The Impact ◽

Lps Treatment ◽

Marrow Cells

Abstract Abstract 2629 The overall objectives of this study are to investigate the impact of inflammatory conditions on hematopoietic stem cell (HSC) maintenance and to elucidate the underlying mechanisms. HSCs are exposed to a variety of inflammatory conditions through life. How these conditions influence the integrity of HSCs is a fundamental issue of clinical importance but it is poorly understood. Equally unknown is the molecular regulation of HSC maintenance during inflammatory. In this context, our focus is on the role of basic helix-loop-helix (bHLH) proteins, which include transcription activators such as E2A proteins and their inhibitors including Id proteins. We and others have shown that these regulators are involved in normal hematopoiesis such as stem cell function and lineage specific differentiation. Recently, we have obtained evidence to suggest that signaling through Toll-like receptors (TLRs), which is closely linked to inflammation, causes down-regulation of E2A function by stimulating Id1 expression. Therefore, we hypothesize that inflammatory conditions causes down-regulation of E protein function, which disturbs the quiescence of long-term (LT)-HSC, leading to stem cell exhaustion over time. To test this hypothesis, we induced chronic inflammation in wild type and Id1-/- mice by daily injection of 1 mg of LPS, i.p. for 30 days. Peripheral blood was collected on days 15 and 30 and levels of a panel of inflammatory cytokines were assayed using a Luminex multiplex kit. On day 15, dramatic increases were found in the levels of IL-10, IL-6, KC and TNFα but not IFN-γ, IL12-p70 and IL-1β. Interestingly, levels of IL-6 and TNFα were significantly lower in Id1-/- mice compared to wild type mice. By day 30 of LPS treatment, levels of these cytokines returned to the levels in animals without LPS injection. These results suggest that this chronic LPS treatment indeed elicited an inflammatory response that included transient elevation of inflammatory cytokines. Whether secretion of these cytokines has any direct effects on HSCs remains to be determined. To measure HSC activity in these LPS-treated mice, we performed serial bone marrow transplant assays. Lin−Sca-1+c-kit+ (LSK) stem/progenitor cells were isolated from wild type or Id1-/- mice treated with or without LPS. These cells were transplanted into lethally irradiated CD45.1+ recipients along with equal numbers of YFP-expressing LSK as competitors. Six weeks later, cohorts of mice were sacrificed and bone marrow cells were collected. Pooled whole bone marrow cells within each cohort were injected into lethally irradiated secondary recipients. Secondary recipients were sacrificed 8 and 16 weeks post transplant. For assessment of primary and secondary engraftment, bone marrow cells were examined for expression of donor and lineage specific markers. Robust engraftment was observed in primary or secondary recipients. Donor derived cells were then gated for YFP− and YFP+ cells, which separate cells originated from tester and competitor LSK, respectively. While YFP− and YFP+ cells engrafted equivalently in primary recipients transplanted with cells treated with or without LPS, LPS treatment of wild type mice caused a great disparity in secondary recipients. In contrast, HSC in Id1-/- mice did not appear to be affected by the same treatment even though HSCs in Id1 deficient mice are normally lower in numbers and activities as we previously reported. These results suggest that chronic inflammation diminishes the LT-stem cell activity and this may involve the up-regulation of Id1 expression. To investigate the underlying mechanism, we performed label retaining assays to examine the quiescence of LT-HSCs. We found that BrdU-labeling in HSCs was 2-fold lower in mice treated with LPS compared to the untreated controls, suggesting that treatment with LPS promoted the cycling of HSCs, thus impairing their stem cell function. Taken together, our study illustrates that chronic inflammation has a detrimental effect on LT-stem cell activity. Although HSCs have an enormous capability to repopulate the bone marrow by compensatory proliferation, pro-longed inflammation could eventually lead to stem cell exhaustion and seriously compromise hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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