CXCR4 Drives Lympho-Myeloid Fate of Hematopoietic Progenitors Via mTOR and Mitochondrial Metabolic Pathways

Abstract Blood production is a tightly regulated process that starts with hematopoietic stem cells (HSCs). In adults, HSCs are unique in their capacity to self-renew and replenish the entire blood system through production of a series of increasingly committed progenitor cells within the bone marrow (BM) microenvironment. HSCs form a rare, quiescent population that displays a metabolism skewed towards anaerobic glycolysis at the expense of mitochondrial oxidative phosphorylation (OXPHOS) to preserve its quiescent state and long-term reconstitution capacity. However, when HSCs differentiate, they undergo a metabolic switch from anaerobic glycolysis to mitochondrial OXPHOS, a process that is in part mediated by the metabolic sensor mTOR. It is well-established that HSCs in the BM adapt the production of myeloid and lymphoid cells depending on the needs of the body and that metabolic plasticity is a critical driver of HSC fate decisions. This has never been assessed for multipotent progenitors (MPPs) which constitute the stage at which the major divergence of lymphoid and myeloid lineages occurs. In mice, common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs) are generated from phenotypically and functionally distinct subpopulations of lineage-biased MPPs, i.e. MPP2 and MPP3 are reported as distinct myeloid-biased MPP subsets that operate together with lymphoid-primed MPP4 to control blood leukocyte production. This question is thus of paramount importance to understand how the lympho-myeloid specification process is regulated. Signaling by the G protein-coupled receptor CXCR4 on MPPs in response to stimulation by its natural ligand, the chemokine CXCL12, produced by BM perivascular stromal cells constitutes a key pathway through which the niches and MPPs communicate. However, the mechanisms whereby CXCR4 signaling regulates MPP specification are still unknown. We addressed this point using BM samples of patients with WHIM Syndrome (WS), a rare immunodeficiency caused by inherited heterozygous autosomal gain-of-CXCR4-function mutations affecting desensitization of CXCR4 and characterized by chronic lympho-neutropenia, as well as a unique WS mouse model which phenocopies severe pan-leukopenia. We unraveled myeloid skewing of the hematopoietic stem and progenitor cell (HSPC) compartment in BM of patients with WS and of WS mice. This relied on CXCR4 signaling strength that controls the output of the lymphoid and myeloid lineages by coordinating the composition and molecular identity of the MPP compartment. The fate of the lymphoid-biased MPP4 subset was central in such a process. Indeed, CXCR4 signaling termination was required for efficient generation and maintenance of the MPP4 pool, while regulating intrinsically their cell cycle status and lymphoid-myeloid gene landscape. In fact, we demonstrated for the first time that enhanced mTOR signaling, accumulation of damaged mitochondria and overactive OXPHOS-driven metabolism promoted cell-autonomous molecular changes that reprogram mutant MPP4 away from lymphoid differentiation. Consistent with this, in vivo chronic treatment with the CXCR4 antagonist AMD3100/Plerixafor or the mTOR inhibitor Rapamycin normalized mitochondrial metabolism and MPP4 differentiation. Thus, our study shows that CXCR4 signaling acts through the mTOR pathway as an essential gatekeeper for integrity of the mitochondrial machinery, which in turn controls lymphoid potential of MPP4. Disclosures No relevant conflicts of interest to declare.

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Overexpression of the TGF-Beta Modulator .Bambi Promotes Hematopoetic Stem Cell Proliferation.

Blood ◽

10.1182/blood.v114.22.1446.1446 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1446-1446

Author(s):

Rui Mao ◽

Olga Sirin ◽

Margaret Goodell

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Receptor Activation ◽

Lymphoid Cells ◽

Quiescent State ◽

Tgf Beta ◽

Hematopoietic Stem

Abstract Abstract 1446 Poster Board I-469 Hematopoietic stem cells (HSC) normally reside in a quiescent state in the bone marrow. During times of stress, HSCs are activated to begin differentiation and self-renewal, replenishing the supply of myeloid and lymphoid cells present in the blood. The mechanisms regulating this rapid activation have not been fully elucidated. We previously identified the TGF-beta modulator Bambi (BMP and activin membrane-bound inhibitor) to be upregulated four-fold in HSCs compared to differentiated cells. Bambi codes for a transmembrane pseudoreceptor that inhibits TGF-beta receptor activation. Since TGF-beta signaling has been established to be important for induction of HSC quiescence as well as cell-cycle inhibition in long-term progenitors, we hypothesize that Bambi may play an important role in the regulation of HSCs. Using a retroviral vector, we overexpressed Bambi in bone marrow cells. Overexpression of Bambi resulted in increased colony-formation in vitro when compared to control cells. Furthermore, transduced cells expressed higher levels of the cell-cycle marker Ki-67, indicating a greater proportion of cells in active stages of the cell cycle. To verify the results of these assays in vivo, bone marrow overexpressing Bambi was transplanted into lethally irradiated recipient mice. Bambi-overexpressing cells demonstrated a higher level of engraftment in all lineages than control cells at several time points, which confirms the previous in vitro data suggesting greater cell cycle activity. Moreover, we identified the pathway through which Bambi acts by monitoring the levels of phosphorylated Smad2 (pSmad2), a downstream target of TGF-beta. Overexpression of Bambi resulted in a distinctly lower level of pSmad2, which explains the cell-cycle effects seen in vivo and in vitro. These studies show that Bambi functions to promote HSC proliferation and a probable mode of action in HSCs is through decreased pSmad2 levels from inhibition of the TGF-beta pathway. Bambi has been shown to be upregulated in certain leukemias, and a more complete understanding of the mechanism through which Bambi acts will provide better opportunities for therapeutic innovation. This research was graciously funded by an NIH grant and the ASH Trainee Research Award. Disclosures No relevant conflicts of interest to declare.

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Human CD34+ Cells Rapidly Mobilized by AMD3100 Repopulate NOD/SCID Mice with Equivalent Efficiency as CD34+ Cells Mobilized by G-CSF.

Blood ◽

10.1182/blood.v104.11.1186.1186 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1186-1186

Author(s):

David A. Hess ◽

Jesper Bonde ◽

Timothy P. Craft ◽

Louisa Wirthlin ◽

John F. DiPersio ◽

...

Keyword(s):

Mononuclear Cells ◽

Paired Comparison ◽

Drug Clearance ◽

Scid Mice ◽

Lymphoid Cells ◽

Additional Patient ◽

Cxcr4 Antagonist ◽

Post Transplantation ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Interactions between the chemokine receptor CXCR4 and its ligand, stromal derived factor-1, regulate hematopoietic stem cell migration. The CXCR4 antagonist, AMD3100, has recently been shown to rapidly mobilize primitive hematopoietic cells. However, the functional properties of stem and progenitor cells mobilized with this agent are not well characterized. Thus, we directly compared the NOD/SCID repopulating function of CD34+ cells rapidly mobilized (4 hours) by AMD3100 versus CD34+ cells mobilized by 5 days of G-CSF treatment. Healthy, matched sibling donors were leukapheresed after a single injection of 240ug/kg AMD3100, and produced an enrichment of circulating CD34+ cells to 0.6% of the total mononuclear cells (MNC). After 2 weeks of drug clearance, the same donor was mobilized with G-CSF (0.4% CD34+ cells), allowing a paired comparison of the repopulating function of cells mobilized by these two regimens. Total MNC, CD34+ cells (>95% purity), and lineage depleted (Lin−) cells (48–55% CD34+) were isolated and transplanted into NOD/SCID mice at various doses. Injection of 106–107 (approximately 5x103–5x104 CD34+ cells) AMD3100-mobilized MNC resulted in bone marrow (BM) engraftment in 6 of 10 mice, whereas equal doses of G-CSF mobilized MNC engrafted 3 of 10 mice. Higher cell doses (2x107 MNC, approximately 1x105 CD34+ cells) consistently produced engraftment in the BM, spleen, and peripheral blood of all mice, with higher levels of engraftment with AMD3100-mobilized cells (1.8±0.5%) compared to G-CSF-mobilized cells (0.4±0.05%, p<0.05). Similar analyses performed using purified CD34+ cells revealed similar engraftment frequencies for both leukapheresis products. Transplantation of 5x104–105 AMD3100-mobilized CD34+ cells engrafted 4 of 7 mice and G-CSF-mobilized CD34+ cells engrafted 5 of 10 mice. However, transplantation of 5x105 AMD3100-mobilized CD34+ cells consistently resulted in higher engraftment levels compared to G-CSF-mobilized CD34+ cells (3.4±1.1% versus 0.8±0.4% human cells, p<0.05). Multilineage hematopoietic differentiation of transplanted CD34+ cells was similar for AMD3100 and G-CSF-mobilized CD34+ cells, with production of myeloid cells (CD33+, CD14−CD13+CD66abce+/−), monocytes (CD14+), immature B-lymphoid cells (CD19+/−CD20+/−), and primitive repopulating (CD34+CD133+CD38−) cells 7–8 weeks post-transplantation. Similarly, AMD3100 and G-CSF-mobilized Lin− cells produced consistent engraftment in all transplanted mice (n=20), and demonstrated equivalent engraftment levels and multilineage differentiation when directly comparing AMD3100 versus G-CSF-mobilization. Ongoing analysis of additional patient samples will allow direct comparison of these repopulating cells by limiting dilution analysis using Poisson statistics. These preliminary studies indicate that human AMD3100-mobilized CD34+ cells possess at least equivalent repopulating capacity compared to G-CSF mobilized cells, and therefore represent a more rapidly obtainable source of hematopoietic stem cells for clinical transplantation.

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Mobilization Studies in Complement-Deficient Mice Reveal That AMD3100 Mobilization Depends On Complement Cascade Activation and Suggest Involvement of “Membrane Attack Complex - MAC” in Egress of Hematopoietic Stem/Progenitor Cells.

Blood ◽

10.1182/blood.v114.22.367.367 ◽

2009 ◽

Vol 114 (22) ◽

pp. 367-367

Author(s):

Marcin Wysoczynski ◽

HakMo Lee ◽

Rui Liu ◽

Wan Wu ◽

Janina Ratajczak, ◽

...

Keyword(s):

Progenitor Cells ◽

Conflicts Of Interest ◽

Membrane Attack Complex ◽

Classical Pathway ◽

Compensatory Mechanism ◽

Complement Cascade ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Deficient Mice

Abstract Abstract 367 We reported that complement cascade (CC) becomes activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPCs) by immunoglobulin (Ig)-dependent pathway and/or by alternative Ig-independent pathway as seen during G-CSF- or Zymosan mobilization, respectively. As a result, several potent bioactive CC anaphylatoxins (C3 and C5 cleavage fragments) are released that regulate egress of HSPCs (Blood 2003;101,3784; Blood 2004;103,2071; Blood 2005;105,40, Leukemia 2009; in press.). This explains why: i) NOD/SCID and RAG-/- animals that do not activate the Ig-dependent CC classical pathway; ii) C2fB-/- and C3-/- mice that do not activate the classical and alternative CC pathways; and iii) C5-/- mice that do not activate the distal pathway of CC are all poor G-CSF- and/or Zymosan mobilizers. In this study, we evaluated the role of CC in mobilization induced by CXCR4 antagonist AMD3100. We noticed that all CC activation-deficient mice mentioned above, except C5-/- mice, mobilize normally in response to AMD3100 administration. Accordingly, the number of mobilized CD34- SKL cells, leucocytes, and CFU-GM clonogeneic progenitors in mutant mice was similar to wt littermates. More important we observed that AMD3100 mobilization of HSPCs was preceded by a massive egress of leucocytes from BM and that AMD3100 was able to stimulate in these cells i) phosphorylation of MAPKp42/44 and ii) secretion of MMP-9. At the same time, ELISA data to detect CC activation revealed that serum levels of CC cleavage fragments, which were low in the initial phase of AMD3100 mobilization during granulocyte egress, become elevated later during HSPC egress. Thus, our data show that despite a fact that G-CSF and AMD3100 mobilize HSPCs by involving different mechanisms, activation of CC is a common phenomenon occurring during mobilization induced by both compounds. This further supports a pivotal role of CC activation in the egress of HSPCs from BM; however, both compounds activate CC differently. While G-CSF administration initiates CC activation at its proximal C1q-C3 level, AMD3100 induces CC activation at the distal C5 level, pointing to a crucial role of C5 cleavage in executing mobilization. To support this, all mice employed in our studies that display defects in activation of proximal stages of CC (NOD/SCID, RAG, C2fB-/-, and C3-/-) are normal AMD3100 mobilizers. However, C5 is cleavage required for mobilization occurs in the plasma of these animals latter on - directly by proteases released from AMD3100-stimulated granulocytes that egress from the BM as a first wave of mobilized cells. This compensatory mechanism cannot occur from obvious reasons in C5-/- mice. We conclude that AMD3100-directed mobilization similarly as G-CSF-induced one depends on activation of CC; however, AMD3100 in contrast to G-CSF activates CC at distal stages – directly by proteases released from mobilized/activated granulocytes. Cleavage of C5 and release of C5a and desArgC5a create a sinusoid-permissive environment in BM for HSPCs egress. This suggests involvement of both C5 cleavage fragments as well as a potential role of downstream elements of CC activation - membrane attack complex - MAC (C5b-C9) in stem cell mobilization. Therefore, some poor AMD3100 patient responders could possess a defect in activation of the distal steps of CC. Disclosures: No relevant conflicts of interest to declare.

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Innate Lymphoid Cell-Derived IL-22 Mediates Endogenous Thymic Repair Under the Control of IL-23

Blood ◽

10.1182/blood.v118.21.143.143 ◽

2011 ◽

Vol 118 (21) ◽

pp. 143-143

Author(s):

Jarrod A Dudakov ◽

Alan M Hanash ◽

Lauren F. Young ◽

Natalie V Singer ◽

Mallory L West ◽

...

Keyword(s):

Conflicts Of Interest ◽

Ex Vivo ◽

Critical Role ◽

Lymphoid Cells ◽

Thymic Epithelial Cells ◽

Immune Competence ◽

Strong Negative Correlation ◽

Hematopoietic Stem ◽

Stimulation Of

Abstract Abstract 143 Despite being exquisitely sensitive to insult, the thymus is remarkably resilient in young healthy animals. Endogenous regeneration of the thymus is a crucial function that allows for renewal of immune competence following infection or immunodepletion caused by cytoreductive chemotherapy or radiation. However, the mechanisms governing this regeneration remain poorly understood. Thymopoiesis is a highly complex process involving cross-talk between developing thymocytes and their supporting non-hematopoietic stromal microenvironment, which includes highly specialized thymic epithelial cells (TECs) that are crucial for T cell development. IL-22 is a recently identified cytokine predominantly associated with maintenance of barrier function at mucosal surfaces. Here we demonstrate for the first time a critical role for IL-22 in endogenous thymic repair. Comparing IL-22 KO and WT mice we observed that while IL-22 deficiency was redundant for steady-state thymopoiesis, it led to a pronounced and prolonged loss of thymus cellularity following sublethal total body irradiation (SL-TBI), which included depletion of both thymocytes (p=0.0001) and TECs (p=0.003). Strikingly, absolute levels of IL-22 were markedly increased following thymic insult (p<0.0001) despite the significant depletion of thymus cellularity. This resulted in a profound increase in the production of IL-22 on a per cell basis (p<0.0001). These enhanced levels of IL-22 peaked at days 5 to 7 after SL-TBI, immediately following the nadir of thymic cellularity. This was demonstrated by a strong negative correlation between thymic cellularity and absolute levels of IL-22 (Fig 1a). In mucosal tissues the regulation of IL-22 production has been closely associated with IL-23 produced by dendritic cells (DCs) and ex vivo incubation of cells with IL-23 stimulates the production of IL-22. Following thymic insult there was a significant increase in the amount of IL-23 produced by DCs (Fig 1b) resulting in similar kinetics of intrathymic levels of IL-22 and IL-23. We identified a population of radio-resistant CD3−CD4+IL7Ra+RORg(t)+ thymic innate lymphoid cells (tILCs) that upregulate both their production of IL-22 (Fig 1c) and expression of the IL-23R (p=0.0006) upon exposure to TBI. This suggests that they are responsive to IL-23 produced by DCs in vivo following TBI and, in fact, in vitro stimulation of tILCs by IL-23 led to upregulation of Il-22 production by these cells (Fig 1d). We found expression of the IL-22Ra on cortical and medullary TECs (cTECs and mTECs, respectively), and uniform expression across both mature MHCIIhi mTEC (mTEChi) and immature MHCIIlo mTECs (mTEClo). However, in vitro stimulation of TECs with recombinant IL-22 led to enhanced TEC proliferation primarily in cTEC and mTEClo subsets (p=0.002 and 0.004 respectively). It is currently unclear if IL-22 acts as a maturation signal for mTECs, however, the uniform expression of IL-22Ra between immature mTEClo and mature Aire-expressing mTEChi, together with the preferential promotion of proliferation amongst mTEClo and cTEC seem to argue against IL-22 as a maturational signal but rather as promoter of proliferation, which ultimately leads to terminal differentiation of TECs. Of major clinical importance, administration of exogenous IL-22 led to enhanced thymic recovery (Fig. 1e) following TBI, primarily by promoting the proliferation of TECs. Consistent with this, the administration of IL-22 also led to significantly enhanced thymopoiesis following syngeneic BMT. Taken together these findings suggest that following thymic insult, and specifically the depletion of developing thymocytes, upregulation of IL-23 by DCs induces the production of IL-22 by tILCs and regeneration of the supporting microenvironment. This cascade of events ultimately leads to rejuvenation of the thymocyte pool (Fig. 1f). These studies not only reveal a novel pathway underlying endogenous thymic regeneration, but also identify a novel regenerative strategy for improving immune competence in patients whose thymus has been damaged from infection, age or cytoreductive conditioning required for successful hematopoietic stem cell transplantation. Finally, these findings may also provide an avenue of study to further understand the repair and regeneration of other epithelial tissues such as skin, lung and breast. Disclosures: No relevant conflicts of interest to declare.

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“RUNX1-IRES-GFP Knock-in” Mice Lack Expression of Runx1a: A Versatile Tool for Hematopoietic Stem Cell Analysis

Blood ◽

10.1182/blood.v118.21.2329.2329 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2329-2329

Author(s):

Yukiko Komeno ◽

Ming Yan ◽

Shinobu Matsuura ◽

Miao-Chia Lo ◽

James R. Downing ◽

...

Keyword(s):

Body Weight ◽

Conflicts Of Interest ◽

Lymphoid Cells ◽

Hematopoietic Stem ◽

Blood Indices ◽

Cell Counts ◽

Significant Difference ◽

Versatile Tool ◽

Exon 4 ◽

Runx1 Expression

Abstract Abstract 2329 Previously reported “RUNX1-IRES-GFP knock-in mice” (Blood 2004;103:2522) (KI mice) were generated by replacing exon 4 of runx1 gene with cDNA of Runx1b/c from exon 4 to exon 8 followed by IRES-GFP, aiming to evaluate Runx1 expression in specific lineages and developmental stages during adult hematopoiesis. They are phenotypically normal, fertile, and blood indices are normal. GFP intensity correlates with Runx1 expression level, and shows lineage-specific changes during maturation in myeloid, erythroid, and lymphoid cells. However, the behavior in the hematopoietic stem cells (HSCs) had not been carefully examined. Interestingly, we discovered that this knock-in strategy eliminated Runx1a expression. Since Runx1a expression is relatively higher in HSCs than in differentiated cells, we analyzed HSCs in these mice to evaluate its roles in stable and stress hematopoiesis. We found that LSK fraction in bone marrow (BM) was significantly decreased in KI mice compared to wild type (WT) mice (0.043% vs 0.085%, p = 0.001). Among subpopulations in LSK, short-term HSC and multipotent progenitor fractions were significantly decreased (0.024% vs 0.046%, p = 0.003, 0.0021% vs 0.0026%, p = 0.001, respectively). SLAM marker staining using CD150 and CD48 showed similar results. Competitive repopulation assay showed less functional HSCs in KI mice. However, there was no significant difference in recovery of cell counts after single-dose 5-FU intraperitoneal injection (150 mg/kg body weight) or sublethal irradiation (5 Gy), or survival after weekly 5-FU injection. After G-CSF subcutaneous injection (125 μg/kg body weight, twice daily for 5 days), mobilized WBC or neutrophil in PB showed no difference. However, LSK and long-term HSC in PB were significantly less in KI mice (0.078% vs 0.135%, p = 0.010, 0.043% vs 0.092%, p = 0.029, respectively) while those in BM did not show significant difference (increased to 0.295% and 0.346% in KI and WT mice, respectively). In conclusion, Runx1a plays some non-redundant roles in stable hematopoiesis, while it is dispensable for tested stress hematopoiesis. RUNX1-GFP KI mice are a versatile tool to evaluate roles of Runx1a in normal hematopoiesis and leukemogenesis when combined with other genetic modifications. Disclosures: No relevant conflicts of interest to declare.

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Sociology of Normal Stem and Progenitor Cells in CML Niche

Blood ◽

10.1182/blood.v120.21.1234.1234 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1234-1234

Author(s):

Robert S Welner ◽

Giovanni Amabile ◽

Deepak Bararia ◽

Philipp B. Staber ◽

Akos G. Czibere ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mouse Model ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Hematopoietic Progenitor Cells ◽

Quiescent State ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Abstract Abstract 1234 Specialized bone marrow (BM) microenvironment niches are essential for hematopoietic stem and progenitor cell maintenance, and recent publications have focused on the leukemic stem cells interaction and placement within those sites. Surprisingly, little is known about how the integrity of this leukemic niche changes the normal stem and progenitor cells behavior and functionality. To address this issue, we started by studying the kinetics and differentiation of normal hematopoietic stem and progenitor cells in mice with Chronic Myeloid Leukemia (CML). CML accounts for ∼15% of all adult leukemias and is characterized by the BCR-ABL t(9;22) translocation. Therefore, we used a novel SCL-tTA BCR/ABL inducible mouse model of CML-chronic phase to investigate these issues. To this end, BM from leukemic and normal mice were mixed and co-transplanted into hosts. Although normal hematopoiesis was increasingly suppressed during the disease progression, the leukemic microenvironment imposed distinct effects on hematopoietic progenitor cells predisposing them toward the myeloid lineage. Indeed, normal hematopoietic progenitor cells from this leukemic environment demonstrated accelerated proliferation with a lack of lymphoid potential, similar to that of the companion leukemic population. Meanwhile, the leukemic-exposed normal hematopoietic stem cells were kept in a more quiescent state, but remained functional on transplantation with only modest changes in both engraftment and homing. Further analysis of the microenvironment identified several cytokines that were found to be dysregulated in the leukemia and potentially responsible for these bystander responses. We investigated a few of these cytokines and found IL-6 to play a crucial role in the perturbation of normal stem and progenitor cells observed in the leukemic environment. Interestingly, mice treated with anti-IL-6 monoclonal antibody reduced both the myeloid bias and proliferation defects of normal stem and progenitor cells. Results obtained with this mouse model were similarly validated using specimens obtained from CML patients. Co-culture of primary CML patient samples and GFP labeled human CD34+CD38- adult stem cells resulted in selective proliferation of the normal primitive progenitors compared to mixed cultures containing unlabeled normal bone marrow. Proliferation was blocked by adding anti-IL-6 neutralizing antibody to these co-cultures. Therefore, our current study provides definitive support and an underlying crucial mechanism for the hematopoietic perturbation of normal stem and progenitor cells during leukemogenesis. We believe our study to have important implications for cancer prevention and novel therapeutic approach for leukemia patients. We conclude that changes in cytokine levels and in particular those of IL-6 in the CML microenvironment are responsible for altered differentiation and functionality of normal stem cells. Disclosures: No relevant conflicts of interest to declare.

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Osteocytes Embedded Inside Bone Are Essential for G-CSF-Induced Hematopoietic Stem/Progenitor Mobilization

Blood ◽

10.1182/blood.v118.21.721.721 ◽

2011 ◽

Vol 118 (21) ◽

pp. 721-721 ◽

Cited By ~ 2

Author(s):

Noboru Asada ◽

Yoshio Katayama ◽

Mari Sato ◽

Kentaro Minagawa ◽

Kanako Wakahashi ◽

...

Keyword(s):

Bone Cells ◽

Conflicts Of Interest ◽

Morphological Changes ◽

Fibroblast Growth Factor 23 ◽

Bone Matrix ◽

Fold Increase ◽

Nerve Fibers ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Endosteal Niche

Abstract Abstract 721 Hematopoietic stem/progenitor cells (HSPCs) are released from the bone marrow (BM) to the circulation by granulocyte-colony stimulating factor (G-CSF) via sympathetic nervous system (SNS)-mediated osteoblast suppression (Katayama et al. Cell 2006). We further elucidated that vitamin D receptor is essential for this neuronal control of endosteal niche (Kawamori et al. Blood 2010). Osteoblasts are known to adopt three fates: die by apoptosis, become bone-lining cells, or become embedded in osteoid and then in mineralized bone matrix to terminally differentiate into osteocytes, which constitute more than 95% of bone cells. Osteocytes have been shown to control the functional balance between osteoblast and osteoclast via mechanotransduction. In order to address the role of bone-embedded osteocytes in HSPCs niche function, we first quantified mRNA expression of bone-related genes in the femur of wild-type (WT) mice to examine if osteocytic function changes during G-CSF treatment (125μg/kg/dose, 8 divided doses, every 12 hours). Whereas markers relating to osteoblast function, osteocalcin and osteopontin, started to decrease late at 6 doses of G-CSF administration when mild mobilization of HSPCs had occurred, osteocyte-specific genes, including neuropeptide y, SOST, MEPE, E11/gp38 and Phex, were rapidly suppressed at 1 dose when no mobilization was observed. These data suggest that osteocytes respond to G-CSF with altered gene expression much earlier than osteoblasts. Further, the number and thickness of osteocyte projections extending toward the endosteal surface were markedly reduced, as assessed by fluorescently labeled phalloidin, at 8 doses of G-CSF treatment when full mobilization was achieved; these morphological changes were observed specifically in newly-embedded osteoid osteocytes, but not in mature osteocytes embedded deep inside mineralized bone. These findings suggest that osteoid osteocytes may sense the signal triggered by G-CSF. We confirmed the presence of β2-adrenergic receptor in osteoid osteocytes and tyrosine hydroxylase-positive nerve fibers in the vicinity by immunofluorecence staining, suggesting that osteoid osteocytes may be regulated by SNS. To directly address osteocyte involvement in G-CSF-induced mobilization, we utilized a transgenic (TG) mice in which inducible and specific ablation of osteocytes is achieved through targeted expression of diphtheria toxin (DT) receptor under DMP-1 promoter. A single injection of DT in TG mice generates “osteocyte-less (OL)” mice. We found that mobilization by G-CSF was drastically impaired in OL mice for progenitors (CFU-Cs, mean±SEM, WT vs Tg: 1673±271 vs 242±94/ml blood, n=6-13, p<0.01; lineage-Sca-1+c-kit+ (LSK) cells, WT vs Tg: 6878±1209/ml vs 1763±502/ml, n=6-13, p<0.01) and stem cells (repopulating units at 4 months, WT vs Tg: 2.5±0.7 vs 0.5±0.2, n=6-7, p<0.05), while the OL BM showed normal HSPC number. The levels of CXCL12 mRNA and protein in BM and bone were markedly decreased during G-CSF treatment even in OL mice despite the mobilization defect, and a CXCR4 antagonist AMD3100 induced mobilization normally in the absence of osteocytes. Thus, osteocytes embedded within the bone are indispensable for G-CSF-induced mobilization through a CXCL12-independent mechanism. Although most of bone-related genes exhibited drastic decreases following G-CSF treatment, we found that fibroblast growth factor 23 (fgf23) mRNA displayed a 4-fold increase at 6 doses of G-CSF. FGF23 is mainly produced by osteocytes and Klotho is an obligate coreceptor for FGF23 to bind and activate FGF receptors. Since we confirmed that klotho hypomorphic (kl/kl) mice showed remarkably disrupted osteocyte network, we injected G-CSF into these mice. As we expected, G-CSF induced virtually no mobilization in kl/kl mice while the number of HSPCs in the BM remained comparable to control mice. Collectively, our results demonstrate a novel function of bone-embedded osteocytes as a critical regulator of HSPC trafficking perhaps by controlling the endosteal niche and establish the important physiologic function of skeletal tissue for hematopoietic microenvironment. Disclosures: No relevant conflicts of interest to declare.

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Hhex Is a Critical Gene In The Development Of Normal and Malignant Lymphoid Cells

Blood ◽

10.1182/blood.v122.21.3788.3788 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3788-3788

Author(s):

Charnise Goodings ◽

Stephen B. Smith ◽

Elizabeth Mathias ◽

Elizabeth Smith ◽

Rati Tripathi ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Transgenic Mice ◽

Conflicts Of Interest ◽

Lymphoid Cells ◽

Conditional Knockout ◽

Hematopoietic Stem ◽

Direct Transcriptional Target ◽

Cell Counts ◽

Transcriptional Target

Abstract Hematopoietically expressed homeobox (Hhex) is a T-cell oncogene. It is frequently deregulated in murine retroviral insertional mutagenesis screens and its enforced expression induces T-cell leukemia in bone marrow transduction and transplantation experiments. We discovered that HHEX is a direct transcriptional target of an LIM domain Only-2 (LMO2)-associated protein complex. HHEX clusters with LMO2-overexpressing T-ALLs and is especially overexpressed in Early T-cell Precursor (ETP) – ALL where it is a direct transcriptional target of LMO2. To further understand Hhex's function, we induced a conditional knockout in floxed Hhex mice with the Vav-iCre transgene. Mice were viable and showed normal blood cell counts with highly efficient deletion of Hhex in all hematopoietic tissues. Thymocytes from conditional knockouts showed a normal pattern of development. Most impressively, Hhex conditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2 transgenic mice (figure 1). Hhex conditional knockouts (Hhex cKOs) also had a significant decrease in mature B cells in the spleen and bone marrow. Interestingly, hematopoietic stem and progenitor cells plated on OP9-GFP or OP9-DL1 stromal cells showed proliferative defects and incomplete differentiation towards both B and T lineage. Also under stress conditions such as sublethal irradiation and competitive bone marrow transplants, Hhex conditional knockouts show a marked defect in both B and T lineages but an increase in early progenitor populations. Our experiments show that Hhex is a critical transcription factor in lymphoid development and in LMO2-induced T-ALL.Figure 1Hhex conditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2 transgenic miceFigure 1. Hhex conditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2 transgenic mice Disclosures: No relevant conflicts of interest to declare.

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Zinc Deficiency In Patients Adults Undergoing To Hematopoietic Stem Cell Post-Transplantation

Blood ◽

10.1182/blood.v122.21.5500.5500 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5500-5500

Author(s):

Andrea Z Pereira ◽

Silva Bernardo Juliana ◽

Silvia MF Pivocari ◽

Marcia Tanaka ◽

Barrere N Ana Paula ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Zinc Deficiency ◽

Conflicts Of Interest ◽

Growth Failure ◽

Serum Zinc ◽

Serum Levels ◽

The Body ◽

Post Transplantation ◽

Hematopoietic Stem

Abstract Introduction Zinc is an important trace mineral for the body, being a cofactor of enzymes responsible for the synthesis of nucleic acids, and maintaining the immune system. Symptoms of zinc deficiency, such as alopecia, diarrhea, skin rash and growth failure, can be confused with those of hematopoietic stem cell post-transplant (HSCT). Some studies show that zinc supplementation, with the purpose of increase of its serum level, reducing the incidence of mucositis, xerostomia, pain and loss of taste. Zinc deficiency is reported in children with leukemia, but there are very few studies in adults. Material and methods 45 adult patients were evaluated, 22 women and 23 men, mean age 49 ± 16 years-old, undergoing HSCT in Hospital Israelita Albert Einstein during the period of 1 year (2012-2013). The serum zinc level, whose average was 69 ± 16 mg/dl, was evaluated in the first day of hospitalization for HSCT and no patient took zinc supplements before it. Results 48% of patients had zinc deficiency, being most prevalent in patients > 60 years, which had 60%. There was no difference between the sexes. Conclusion Some studies believe that zinc, will be a very important agent in transplant medicine, due to its action on the improvement of the severity of mucositis induced by chemotherapy in patients with leukemia. In our study we found a high prevalence of zinc deficiency in adults and the elderly. Therefore, the assessment of zinc serum levels should be considered in patients submitted to HSTC, as a purpose of treatment and improvement of complications related to it. Disclosures: No relevant conflicts of interest to declare.

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Identification of a Developmentally-Restricted Hematopoietic Stem Cell That Gives Rise to Innate-like Lymphocytes

Blood ◽

10.1182/blood.v124.21.4302.4302 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4302-4302

Author(s):

Anna E Beaudin ◽

Scott W. Boyer ◽

Gloria Hernandez ◽

Camilla E Forsberg

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Lineage Tracing ◽

Lymphoid Cells ◽

Gfp Expression ◽

Hematopoietic Stem

Abstract The generation of innate-like immune cells distinguishes fetal hematopoiesis from adult hematopoiesis, but the cellular mechanisms underlying differential cell production during development remain to be established. Specifically, whether differential lymphoid output arises as a consequence of discrete hematopoietic stem cell (HSC) populations present during development or whether the fetal/neonatal microenvironment is required for their production remains to be established. We recently established a Flk2/Flt3 lineage tracing mouse model wherein Flk2-driven expression of Cre recombinase results in the irreversible switching of a ubiquitous dual-color reporter from Tomato to GFP expression. Because the switch from Tom to GFP expression in this model involves an irreversible genetic excision of the Tomato gene, a GFP+ cell can never give rise to Tom+ progeny. Using this model, we have definitively demonstrated that all functional, adult HSC remain Tomato+ and therefore that all developmental precursors of adult HSC lack a history of Flk2 expression. In contrast, adoptive transfer experiments of Tom+ and GFP+ fetal liver Lin-cKit+Sca1+ (KLS) fractions demonstrated that both Tom+ and GFP+ fetal HSC support serial, long-term multilineage reconstitution (LTR) in irradiated adult recipients. We have therefore identified a novel, developmentally restricted HSC that supports long-term multilineage reconstitution upon transplantation into an adult recipient but does not normally persist into adulthood. Developmentally-restricted GFP+ HSC display greater lymphoid potential, and regenerated both innate-like B-1 lymphocytes and Vg3-expressing T lymphocytes to a greater extent than coexisting Tom+ FL and adult HSC. Interestingly, whereas developmental regulation of fetal-specific B-cell subsets appears to be regulated cell-instrinsically, as fetal HSC generated more innate-like B-cells than adult HSC even within an adult environment, T-cell development may be regulated both cell intrinsically and extrinsically, as both the cell-of-origin and the fetal microenvironment regulated the generation of innate-like T-cells. Our results provide direct evidence for a developmentally restricted HSC that gives rise to a layered immune system and describes a novel mechanism underlying the source of developmental hematopoietic waves. As early lymphoid cells play essential roles in establishing self-recognition and tolerance, these findings are critical for understanding the development of autoimmune diseases, allergies, and tolerance induction upon organ transplantation. Furthermore, by uncoupling self-renewal capacity in situ with that observed upon transplantation, our data suggests that transplantation- and/or irradiation-induced cues may allow for the engraftment of developmental HSC populations that do not normally persist in situ. As LTR upon transplantation has served as the prevailing definition of adult HSC origin during development, our data challenge the current conceptual framework of adult HSC origin. Disclosures No relevant conflicts of interest to declare.

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