scholarly journals An Irradiation-Altered Bone Marrow Microenvironment Impacts Anabolic Actions of PTH

Endocrinology ◽  
2011 ◽  
Vol 152 (12) ◽  
pp. 4525-4536 ◽  
Author(s):  
A. J. Koh ◽  
C. M. Novince ◽  
X. Li ◽  
T. Wang ◽  
R. S. Taichman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Formation ◽  
Bone Marrow Microenvironment ◽  
Fibroblast Growth Factor 2 ◽  
Mrna Levels ◽  
Hematopoietic Stem ◽  
Cell Recruitment ◽  
Anabolic Activity ◽  
Marrow Cellularity

PTH stimulates bone formation and increases hematopoietic stem cells through mechanisms as yet uncertain. The purpose of this study was to identify mechanisms by which PTH links actions on cells of hematopoietic origin with osteoblast-mediated bone formation. C57B6 mice (10 d) were nonlethally irradiated and then administered PTH for 5–20 d. Irradiation reduced bone marrow cellularity with retention of cells lining trabeculae. PTH anabolic activity was greater in irradiated vs. nonirradiated mice, which could not be accounted for by altered osteoblasts directly or osteoclasts but instead via an altered bone marrow microenvironment. Irradiation increased fibroblast growth factor 2, TGFβ, and IL-6 mRNA levels in the bone marrow in vivo. Irradiation decreased B220 cell numbers, whereas the percent of Lin−Sca-1+c-kit+ (LSK), CD11b+, CD68+, CD41+, Lin−CD29+Sca-1+ cells, and proliferating CD45−Nestin+ cells was increased. Megakaryocyte numbers were reduced with irradiation and located more closely to trabecular surfaces with irradiation and PTH. Bone marrow TGFβ was increased in irradiated PTH-treated mice, and inhibition of TGFβ blocked the PTH augmentation of bone in irradiated mice. In conclusion, irradiation created a permissive environment for anabolic actions of PTH that was TGFβ dependent but osteoclast independent and suggests that a nonosteoclast source of TGFβ drives mesenchymal stem cell recruitment to support PTH anabolic actions.

scholarly journals Cell-nonautonomous function of Id1 in the hematopoietic progenitor cell niche

Blood ◽  
2009 ◽  
Vol 114 (6) ◽  
pp. 1186-1195 ◽  
Author(s):  
Hyung Chan Suh ◽  
Ming Ji ◽  
John Gooya ◽  
Michael Lee ◽  
Kimberly D. Klarmann ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cell ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Cytokine Levels ◽  
Cell Niche ◽  
Marrow Cellularity

Abstract Development of hematopoietic stem cells (HSCs) and their immediate progeny is maintained by the interaction with cells in the microenvironment. We found that hematopoiesis was dysregulated in Id1−/− mice. Although the frequency of HSCs in Id1−/− bone marrow was increased, their total numbers remained unchanged as the result of decreased bone marrow cellularity. In addition, the ability of Id1−/− HSCs to self-renew was normal, suggesting Id1 does not affect HSC function. Id1−/− progenitors showed increased cycling in vivo but not in vitro, suggesting cell nonautonomous mechanisms for the increased cycling. Id1−/− HSCs developed normally when transplanted into Id1+/+ mice, whereas the development of Id1+/+ HSCs was impaired in Id1−/− recipients undergoing transplantation and reproduced the hematologic features of Id1−/− mice, indicating that the Id1−/− microenvironment cannot support normal hematopoietic development. Id1−/− stromal cells showed altered production of cytokines in vitro, and cytokine levels were deregulated in vivo, which could account for the Id1−/− hematopoietic phenotypes. Thus, Id1 is required for regulating the hematopoietic progenitor cell niche but is dispensable for maintaining HSCs.

Hijacking the Niche: The Role of Stromal Osteopontin in the Induction of Leukemic Dormancy within the Bone Marrow Microenvironment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 754-754
Author(s):  
Benjamin Boyerinas ◽  
Ali Ekrem Yesilkanal ◽  
Andrea Pontier ◽  
Dorothy A. Sipkins
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
In Vivo Imaging ◽  
Tumor Burden ◽  
Bone Marrow Microenvironment ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Bone Marrow Biopsies ◽  
Stromal Microenvironment

Abstract Abstract 754 Introduction: Acute lymphoblastic leukemia (ALL) is a treatable malignancy where initial induction chemotherapy achieves clinical remission in the majority of patients. Relapsed disease, however, occurs in many patients and is significantly more difficult to treat. The majority of relapsed cases are a result of minimal residual disease (MRD) that persists within the bone marrow (BM) after initial chemotherapy. Our evolving knowledge of the importance of the host microenvironment in tumor progression suggests that the stromal microenvironment can protect leukemic cells from chemotherapeutic assault, and that inhibiting the supportive relationship between leukemic blasts and the bone marrow microenvironment (BMM) will provide novel therapeutic opportunities. We therefore aimed to identify and characterize novel stromal signaling mechanisms that retain and support blasts within the malignant BMM. Our preliminary data suggest that osteopontin (OPN), normally secreted by osteoblasts within the marrow, is one such signaling chemokine that is highly upregulated in the leukemic niche. OPN has well-defined roles in both solid tumor metastasis and normal hematopoietic stem cell function within the BMM. Specifically, OPN expression at the endosteal bone surface functions to recruit hematopoietic progenitors to bone where they are induced to become quiescent and maintain long term repopulating potential. We hypothesized that a similar relationship exists between leukemia and OPN resulting in a quiescent population of chemoresistant leukemic blasts at the BM endosteum. Here, we demonstrate that stromal OPN negatively regulates leukemia cell proliferation in the BMM. Methods: A GFP expressing clone of the pre-B ALL cell line Nalm-6 (10 × 106 cells) was engrafted into SCID hosts (6-8 weeks old) via tail vein injection. In vivo imaging was accomplished in live anesthetized mice by reflecting the scalp and imaging the calvarial marrow compartment using real-time multi-photon confocal microscopy. OPN expression in the malignant marrow was imaged by injecting engrafted mice with fluorescently conjugated anti-OPN antibodies 18hrs prior to imaging. For OPN neutralizing experiments, engrafted mice were injected with a cocktail of anti-mouse and anti-human OPN antibodies at a dose of 3 mg/kg. Results: Using PCR, Western blot and ELISA assays, we show that the ALL cell line Nalm-6 expresses OPN and secretes large quantities of OPN into conditioned media. Flow cytometric analysis demonstrates that Nalm-6 also express the cell surface OPN receptors VLA-4 and VLA-5. Furthermore, Nalm-6 cells specifically adhere to OPN in vitro via specific engagement of these integrin receptors. In vivo imaging demonstrates that OPN expression in the BM increases as leukemia progresses and that OPN is highly expressed adjacent to areas of high tumor burden. Specifically, a significant amount of OPN is detected in bony tunnels surrounding the vasculature at the invading tumor front. Using Q-PCR and western analysis, we demonstrate that both host-derived and leukemia-derived OPN are upregulated in malignant BM. In vivo inhibition of the OPN signaling axis in the Nalm-6 xenograft model using neutralizing antibodies directed at both human and murine OPN increased overall tumor burden two-fold as measured by flow cytometry and in vivo imaging (p=0.02, N=7) while simultaneously increasing the Ki-67 positive proliferative tumor population (p=0.029, N=4). Furthermore, IHC analysis of a panel of diagnostic bone marrow biopsies from a diverse cohort of ALL patients demonstrated high OPN expression in the marrow of these patients. Conclusion: Leukemic blasts that have hijacked normal stromal interactions to become quiescent may represent a major source of MRD and patient relapse in ALL. Our data demonstrate that the interaction of leukemic blasts with OPN in the stromal microenvironment reduces the number of cycling blasts and constrains tumor proliferation within the marrow. Current investigations are aimed at combining OPN neutralization with an in vivo model of MRD to determine whether OPN neutralization induces cycling of quiescent blasts, ultimately rendering them sensitive to chemotherapy. The ultimate goal of this work is the development of clinically relevant therapies designed to render leukemic cells more susceptible to chemotherapy by disengaging them from protective interactions with the BM microenvironment. Disclosures: No relevant conflicts of interest to declare.

Hyaluronan Associated with the Bone Marrow Hematopoietic Microenvironment Is Required for the Recruitment and Retention of Hematopoietic Stem and Progenitor Cells In the Bone Marrow.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2593-2593
Author(s):  
Valentina Goncharova ◽  
Shinji Iizuka ◽  
Yu Yamaguchi ◽  
Sophia Khaldoyanidi
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Knockout Mice ◽  
Peripheral Blood ◽  
Bone Marrow Microenvironment ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Hematopoietic Microenvironment ◽  
Ha Synthase

Abstract Abstract 2593 The bone marrow microenvironment regulates a variety of hematopoietic stem cell (HSC) functions, including their recruitment into the marrow following transplantation. However, an insufficient understanding of the biology of the hematopoietic microenvironment reflects gaps in our knowledge of fundamental stem cell biology and remains an obstacle to an optimal therapeutic approach. While the involvement of hyaluronan (HA), one of the major components of the bone marrow ECM, in normal cell and tumor biology is generally appreciated, little is known of how HA contributes to the regulation of the hematopoietic microenvironment. In this study we investigated whether HA contributes to the recruitment of circulating HSCs into marrow. Stimulation of bone marrow cells with HA induced production of chemokines including SDF-1, MIP-1a, MIP-1b, IL-8, eotaxin, CXCL16, RANTES, LIX and MCP-1. HA-induced conditioned media containing these chemokines enhanced SDF-1-mediated HSC transmigration in vitro, whereas HA alone had no effect, suggesting that endogenous HA participates in the recruitment of HSC indirectly, via regulation of the production of chemokines. We next tested the relevance of this finding in vivo. Since HA synthase 2 knockout mice are embryonically lethal, we developed an alternative in vivo model that allows deprivation of HA. Mice were lethally irradiated to eliminate HSCs and to degrade endogenous HA, and injected with 4-MU, an HA synthase inhibitor, to prevent de novo synthesis of HA. Pre-treated mice were transplanted with HSCs, and after 24 hours the bone marrow was assayed by competitive reconstitution. This assay demonstrated that the number of HSCs which migrated into the marrow of mice pretreated with 4MU was significantly lower as compared to control. To further understand the role of endogenous HA in bone marrow microenvironment we used compound mutant mice in which Has1 and Has3, two genes encoding hyaluronan synthase, were knocked out (Has1-/-;Has3-/-). The number of hematopoietic progenitors (HPs) circulating in peripheral blood was significantly increased in the double knockout (dKO) mice as compared to the wild type (WT) mice. This increase in the number of circulating HP was further enhanced in triple Has knockout mice (Prx1-Cre;Has2flox/flox;Has1-/-;Has3-/-). This correlated with the higher number of hematopoietic progenitors in spleens of Has1-/-;Has3-/- double KO mice and Prx1-Cre;Has2flox/flox;Has1-/-;Has3-/- triple KO mice as compared to the WT mice. In summary, our data indicate that low levels of HA correlate with re-distribution of HSCs and HPs from bone marrow into peripheral blood and spleen. This suggests that HA associated with the hematopoietic microenvironment is important for recruitment and retention of HSCs and HPs, confirming that HA plays a critical regulatory role in the hematopoietic microenvironment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals EGF Accelerates Hematopoietic Stem Cell Regeneration Following 5-FU Chemotherapy Via G-CSF Receptor Signaling

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1490-1490
Author(s):  
Sadhna O. Piryani ◽  
Angel Y.F. Kam ◽  
Evelyna G. Kliassov ◽  
Benny J. Chen ◽  
Neil L. Spector ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Growth Factor ◽  
Conflicts Of Interest ◽  
Lethal Dose ◽  
Flow Cytometric Analysis ◽  
Mrna Levels ◽  
Receptor Signaling ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cells (HSCs) reside in specialized bone marrow microenvironments adjacent to endothelial cells (BM ECs). BM ECs regulate both HSC self-renewal and regeneration by expressing soluble growth factors such as epidermal growth factor (EGF). EGF accelerates HSC regeneration following ionizing radiation injury (Doan et al., Nat Med, 2013), though its role in HSC reconstitution after chemotherapy is not yet defined. At 24 hours following administration of 5-fluorouracil (5-FU), the expression of EGF receptor is increased 10-fold (p<0.0001) compared to untreated control mice within ckit+Sca-1+Lineage- (KSL) cells. EGFR expression is preferentially induced 6.8-fold in KSL cells compared to whole bone marrow cells after 5-FU (p=0.01). When C57Bl6 mice are treated with a single injection of 150 mg/kg 5-FU followed by daily injections of EGF for 4 days, they demonstrated preserved BM cellularity (p=0.0003), increased BM EC density (p=0.002), and contained higher levels of donor engraftment at 16-weeks post-secondary transplantation in competitive repopulating assays (mean 21.9% vs 52.2%, p=0.0005) compared to saline-treated control mice. Following a lethal dose of 5-FU, EGF-treated mice displayed 70% survival compared to 20% survival in saline-treated control mice (p=0.04). These results suggest that EGF signaling may accelerate both hematopoietic progenitor and stem cell reconstitution following myelosuppressive and lethal-dose chemotherapy. Since the pro-apoptotic gene Bax is increased 2.8-fold in BM lin- cells following 5-FU compared to cells from untreated mice (p<0.0001), we sought to determine whether chemo-protection of BM ECs via deletion of Baxwould accelerate HSC reconstitution. We employed a genetic mouse model with Cre recombinase to delete Bax specifically in VECadherin-expressing ECs (VECadherinCre;BaxFL/+ and VECadherinCre;BaxFL/FL mice). These mice displayed no differences in hematopoiesis at baseline. When C57Bl6 KSL cells are in non-contact co-cultures with BaxFL/+ BM ECs and treated with 5-fluorodeoxyuridine monophosphate (FdUMP) + EGF for 48 hours, they displayed both increased colony-forming cells (CFCs) and decreased annexin+ cells compared to control cultures (p=0.001 and p=0.03, respectively). Conversely, when C57Bl6 KSL cells are cultured with BaxFL/FL ECs and FdUMP + erlotinib, a tyrosine kinase inhibitor that neutralizes EGFR signaling, they displayed decreased CFCs and increased annexin+ cells compared to control cultures (p=0.001 and p=0.0003, respectively). These data suggest that chemo-protection of BM ECs by deletion of Bax in vivo results in accelerated hematopoietic recovery. Following 5-FU, EGF promoted cell cycling with increased Ki67+ cells and cells in interphase (mean 4.8% vs 16.1%, p=0.03). Moreover, EGF decreased annexin+ cells within the KSL population both in vitro following FdUMP (mean 26.3 % vs 17%, p=0.002) and in vivo following 5-FU (mean 21.5% vs 11.5%, p<0.0001). Concordant with these results, KSL cells treated with FdUMP + EGF displayed lower levels of double-strand DNA (dsDNA) breaks, measured by γH2AX, compared to control cultures (mean 39% vs 16%, p<0.0001). Since EGF can repair dsDNA breaks via non-homologous end joining recombination, we measured the levels of phosphorylation of DNA pk cs. Following 24 hours with FdUMP and 15 minutes with EGF, KSL cells displayed a 4.8-fold increase in phospho-DNA pk cs (T2647) compared to control cultures (p=0.04). Finally, we sought to determine whether EGF could accelerate HSC reconstitution via the granulocyte colony-stimulating factor (G-CSF) receptor. Following 5-FU + EGF, G-CSF-R expression is increased 3.6-fold in mRNA levels (p<0.0001) and by flow cytometric analysis (mean 23.9% vs 42.4%, p=0.003) compared to 5-FU-treated mice. These results suggest that EGF may be a potent HSC growth factor and may exert its effect at least in part via G-CSF receptor signaling. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

scholarly journals Osteoblastic VEGF Coordinates Remodeling of the Hematopoietic Stem Cell Niche

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 772-772 ◽  
Author(s):  
Julianne N. P. Smith ◽  
Corey M Hoffman ◽  
Alexandra N Goodman ◽  
Laura M. Calvi
Keyword(s):  
Bone Marrow ◽  
Blood Vessels ◽  
Progenitor Cell ◽  
Bone Marrow Microenvironment ◽  
Hematopoietic Stem ◽  
Group 3 ◽  
Osteolineage Cells ◽  
Vascular Branching

Abstract Osteoblasts (OBs) and the bone marrow (BM) vasculature constitute hematopoietic stem and progenitor cell (HSC/HSPC) niches. Immature perivascular osteolineage cells enforce HSC quiescence while signals from mature OBs are capable of bone and vascular remodeling. We previously demonstrated that bone anabolic parathyroid hormone (PTH) stimulation increases HSPCs with short and long-term repopulating activity. Based upon the coupled nature of osteogenesis and angiogenesis as well as the multifaceted role blood vessels play in HSC regulation, we investigated effects of PTH on the BM vasculature. Both genetic PTH receptor activation in maturing OBs (Col1caPTH1R) and systemic PTH treatment increased functional blood vessel branching imaged intravitally in the calvaria (14±2 vs 34±3 and 14±2 vs 27±4 branchpoints, n=3-4 mice/group, 3-7 regions analyzed per mouse). These changes were confirmed histologically in long bones, where PTH-induced tortuosity was accompanied by the emergence of α smooth muscle actin (αSMA)+ bone-associated vascular networks, likely small-caliber arterioles, containing red blood cell rouleaux, while control marrow displayed sparse, non-bone-associated αSMA+ vessels. Further, PTH increased morphologically-heterogeneous BM microvessels (167±18 vs 348±39 per section, n=5 mice/group) and endothelial cell (EC) abundance (0.09±0.02% vs 0.2±0.02%, n=4-5 mice/group). VEGF-A and FGF2, known to be important proangiogenic signals in bone, were increased after PTH treatment of osteoblastic cell lines and primary osteolineage cells in vitro, and in bone-associated cells of mice treated in vivo. Because the perivascular milieu is heterogeneous and can support HSC quiescence or activation depending on vessel type and stimuli, we tested whether tuning BM angiogenic responses modulated effects of PTH on HSCs. Treatment with the anti-VEGF-A monoclonal antibody bevacizumab (αVEGF) precluded PTH-induced vascular branching in calvarial BM and reduced pre-established vascular branching in Col1caPTH1R mice (34±3 vs 21±1 branchpoints, n=4 mice/group, 3-5 regions analyzed per mouse), while PTH-induced bone anabolism, EC abundance and bone-associated αSMA+ small-caliber vessels were sustained. Moreover, αVEGF did not change the frequency of CD51+ PDGFRα+ or PDGFRα+ Sca1+ immature mesenchymal cells reported to regulate HSCs. The altered balance of marrow sinusoidal vs arteriolar structures we quantified in PTH + αVEGF-stimulated BM would be expected to improve HSC support based on niche remodeling, therefore we tested the hematopoietic consequences. αVEGF did not alter frequencies of phenotypically-defined HSCs in the BM or mature hematopoietic cells and platelets in the peripheral blood (PB). Remarkably, αVEGF augmented PTH-induced repopulation of the PB in primary BM transplantation (p = 0.0013, n=10 recipients/group) and sustained the repopulating ability (p < 0.0001, n=10 recipients/group) and BM engraftment (~70 fold) of cells in secondary transplantation. Competitive transplantation of HSPCs sorted from PTH + αVEGF-treated mice showed enhanced repopulating ability, confirming niche-mediated improvement of HSPC function. Unbiased analysis of sorted stem and progenitor cells demonstrated that PTH, alone or in combination with αVEGF, broadly reduced multipotent progenitor (MPP) gene expression, including markers of cell proliferation. Notably, microenvironmental activation with PTH alone uniquely decreased a cluster of transcripts associated with hematopoietic differentiation in MPPs, suggesting progenitor cell reeducation by PTH activation versus niche reapportioning by combined PTH and VEGF antagonism. Because PTH also increases FGF2, reported to expand HSCs and stabilize blood vessels, we tested whether FGF signaling is necessary for PTH to establish long-term HSC niches. In vivo FGF receptor 1 inhibition significantly reduced long-term hematopoietic repopulating ability of PTH + αVEGF-treated BM cells in secondary transplantation (p = 0.0046, n = 16-17 mice/group), suggesting VEGF-A and FGF2 have opposing HSC effects in the PTH-activated microenvironment. These data define the HSC niche as a regulatory network, and identify mature OBs as the cellular source of signals that serve to coordinate HSC-supportive niches, demonstrating functional cooperation of the different constituents of the bone marrow microenvironment. Disclosures Off Label Use: Bevacizumab (trade name Avastin, Genentech/Roche) is used by the authors as a strategy to block VEGF-A in the bone marrow microenvironment.. Calvi:Fate Therapeutics: Patents & Royalties.

The Inhibition of the Osteoblast Niche during Hematopoietic Stem Cell Mobilization Is an Indirect Effect Involving Mature Bone Marrow Leukocytes, IL6 and Soluble IL6 Receptor.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1966-1966 ◽  
Author(s):  
Jean-Pierre Levesque ◽  
Ingrid G. Winkler ◽  
Natalie Sims ◽  
Howard Morris ◽  
Paul J. Simmons ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Formation ◽  
Indirect Effect ◽  
Mrna Levels ◽  
Adhesive Interaction ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Kit Ligand ◽  
Osteoblast Function ◽  
Osteocalcin Production

Abstract Mobilization of hematopoietic stem cells (HSC) involves the disruption of 1) the adhesive interaction between VCAM-1 and α4-integrins and 2) the chemotactic interaction between CXCL12 and CXCR4, interactions which are both required for the retention of HSC within the bone marrow (BM). Experiments in mice deficient in neutrophil proteases have shown that while the disruption of the VCAM-1/α4 integrin interaction is entirely due to the proteolytic cleavage of VCAM-1 by proteases released from neutrophils accumulating in mobilized BM, the down-regulation of CXCL12 involves protease-independent mechanisms. We have recently shown that osteoblasts are the main source of CXCL12 in the BM and that both the number of osteoblasts lining the endosteum and bone formation are dramatically reduced during G-CSF administration as reflected by bone pain often experienced by mobilized donors. Consequently, the decreased level of CXCL12 levels in mobilized BM could be due to inhibition of osteoblasts, an essential component of the hematopoietic niche. Quantitative real-time RT-PCR were performed on mouse BM cells to follow osteocalcin mRNA levels. Osteocalcin mRNA dropped 2–3 logs during mobilization induced by either G-CSF or cyclophosphamide showing that the inhibition of osteoblast function is not restricted to G-CSF-induced mobilization. Morphometric analyses of tibia sections showed a quasi disappearance of osteoblasts and osteoid as early as day 2 of G-CSF injection. In humans, we observed a significant reduction of osteocalcin protein concentration in the plasma during mobilization induced by either G-CSF alone, G-CSF+ KIT ligand or IL3+GM-CSF, showing that in both humans and mice this effect is not restricted to G-CSF. In cultures of purified human osteoblasts, neither G-CSF, KIT ligand, IL3 nor GM-CSF inhibited osteocalcin production demonstrating that inhibition of osteoblast function is not a direct effect of these cytokines. In parallel experiments, addition of differentiated BM CD34− leukocytes to osteoblast cultures, resulted in a dose-dependant inhibition of osteocalcin production showing that the effect is mediated by mature leukocytes. Since IL6 and soluble IL6 receptor (sIL6R) are important mediators of bone formation, we tested these two cytokines on purified osteoblasts and found that the combination of IL6+sIL6R was a potent inhibitor of osteocalcin production while these cytokines had no effect when used alone. Furthermore, we find cocultures of osteoblasts and BM leukocytes results in a 30-fold increase in IL6 production compared to monocultures of osteoblasts or BM leukocytes. Finally, in humans, plasma concentration of sIL6R is significantly increased during HSC mobilization and this increase is significantly correlated with the number of circulating CFU-GM. Taken together, these data indicate that the inhibition of osteoblast function during HSC mobilization is an indirect effect involving mature BM leukocytes, IL6 and sIL6R.

Leukemia Stem Cells Are Resistant to In Vivo, Cell Non-Autonomous Wnt Inhibition.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1025-1025
Author(s):  
Steven W. Lane ◽  
Cristina Lo Celso ◽  
Stephen M Sykes ◽  
Sebastian Shterental ◽  
Mahnaz Paktinat ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Wnt Signaling ◽  
Bone Marrow Microenvironment ◽  
Leukemia Stem Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Dkk1 Expression ◽  
Hsc Niche

Abstract Abstract 1025 Poster Board I-47 Acute myeloid leukemia (AML) initiating cells reside within and utilize the bone marrow microenvironment, as a sanctuary to evade chemotherapy and to maintain self-renewal. Following treatment, these leukemia stem cells (LSC) re-emerge and reconstitute disease, leading to relapse. The canonical Wnt signaling pathway is frequently dysregulated in LSC and recent data indicates that Dkk1 (a potent endogenous Wnt inhibitor) may have a therapeutic role in treating AML. Microenvironment specific Dkk1 expression inhibits hematopoietic stem cell (HSC) Wnt and extinguishes HSC self-renewal in vivo, identifying the Wnt pathway as essential in normal HSC-niche homeostasis. We investigated the importance of bone marrow microenvironment Wnt signaling in LSC survival. AML was generated using retroviral transduction of murine bone marrow with the MLL-AF9 fusion oncogene. We then assessed the potential for niche-directed Wnt inhibition of LSC using 2.3kbColl1alpha-Dkk1 transgenic mice in which Dkk1 expression is restricted to osteoblasts. AML was observed in the Dkk1 or wild type mice with similar disease latency and phenotype. AML was also observed in secondary transplant recipients, although there was a reduction of LSC (linlowcKithighSca-1-FcGRII/III+CD34+) derived from Dkk1 mice (LSC frequency 2.8% WT vs 1.6% Dkk1, p<0.05), correlating with a subtle prolongation in disease latency (n=15, 20 days WT vs. 24 days Dkk1, p<0.001). To determine the status of Wnt signaling in MLL-AF9 AML, we generated AML in bone marrow derived from TOPGal reporter mice that harbor a Tcf/Lef responsive promoter with a LacZ reporter, and quantified LacZ expression or galactosidase protein levels. Wnt activation was increased following transformation of bone marrow with MLL-AF9 (relative TOPGal expression 1.35 empty vector vs 2.58 MLLAF9, p=0.03). To assess the effects of osteoblast-restricted Dkk1 expression in vivo, Wnt signaling was measured in LSC purified by high-speed multiparameter flow cytometry. Reporter activity (fluorescein di-β-D-galactopyranoside (FDG), Invitrogen) was unchanged in LSC from WT or Dkk1 recipients (Median fluorescent intensity 552 vs 542, p=0.85), indicating that, in contrast with normal HSC, Wnt signaling in LSC is relatively resistant to Dkk1 expression in the niche. To better understand the mechanism of LSC resistance to Dkk1, we examined the homing and micro-localization of LSC in vivo using live, 3 dimensional two photon-confocal hybrid imaging of the bone marrow microenvironment. LSC proliferate with similar kinetics in Dkk1 or WT recipients (proliferating fraction 57.7% WT vs 50.3% Dkk1 LSC p=0.48). However, when compared to HSC, LSC home with less affinity to osteoblasts and may escape the effects of osteoblast specified Dkk1 expression through residence in a niche that is physically distant from endosteum (Median distance to osteoblast 18um WT vs 20.6um Dkk1 LSC, p=0.13). Taken together, these data indicate that MLL-AF9 LSC can escape the normal HSC-niche homeostatic constraints regulated by Wnt, an observation that may have important therapeutic implications. Disclosures: Scadden: Fate Therapeutics: Consultancy. Gilliland:Merck: Employment.

Non-Invasive Multimodal/Multi-Scale Imaging of Bone Marrow Microenvironment Reveals Structural and Functional Heterogeneity in Different Bone Marrow Compartments: Functional Implications On Mouse and Human Hematopoietic Stem Cell Niches in Health and Disease

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 25-25
Author(s):  
Francois Lassailly ◽  
Katie Foster ◽  
Lourdes Lopez-Onieva ◽  
Erin Currie ◽  
Dominique Bonnet
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endochondral Ossification ◽  
Bone Marrow Microenvironment ◽  
Hematopoietic Stem ◽  
Non Invasive ◽  
Health And Disease ◽  
Human Hscs ◽  
Stem Cell Niches

Abstract Abstract 25 Introduction: Hematopoietic stem cells (HSCs) reside in specialized bone marrow (BM) microenvironments called stem cell niches (HSCNs). Two types of HSCNs have been reported, involving osteoblasts (osteoblastic niche) and blood sinusoids (vascular niche). Various hematopoietic and non-hematopoietic cell types are contributing to the regulation of HSCs and HSCNs, however, the bone marrow microenvironment has not yet been precisely characterized and the fine localization, composition and regulation of the niches remain highly controversial. Intravital microscopy of the calvarium (IVMC) is the only non-invasive option for high resolution imaging of bone marrow HSCNs. However it is unclear if IVMC provides data representative of all BM compartments. Indeed it has been shown that endochondral ossification, the process used for long bones growth, is required for the formation of HSCNs. By contrast, the calvarium is a flat bone which develops through intra-membranous ossifications, therefore questioning the presence of HSCNs in this bone (Chan CK et al, Nature 2009). Methods: Combined confocal and multiphoton excitation intravital microscopy was used for multiparametric observation of live bone marrow in intact bones after in-vivo contrasting procedures. Bioluminescence imaging was used to quantify the systemic trafficking and proliferation of murine HSCs and human leukemia. Flow cytometry and histology were used to sort specific populations of cells, analyze the frequency of mouse and human stem cells or leukemic cells at steady state or after transplantation, quantify perfusion efficiency and hypoxia and cross-validate in-vivo imaging procedures. Results: Thanks to advanced imaging modalities we realized a thorough study of potential niche markers and HSCs distribution at homeostasis and during hematopoietic reconstitution in calavia, epiphyses and diaphyses. We report important heterogeneity between these compartments in terms of bone remodelling activity (BRA) and blood vessel fraction (BVF). Although BVF was surprisingly high in any compartment, including in very close proximity to any endosteal surface, we found that compartments displaying the highest BVF and BRA were preferentially seeded and engrafted following mouse and human HSC transplantation. Unexpectedly, the macro-anatomical distribution of mouse and human HSCs at steady state is homogeneous across the skeleton and independent of these 2 markers. These data suggest the existence of “reconstituting niches” which would be distinct from “homeostatic niches”. Importantly, this study provides the first evidence that both types of niches are fully functional in the calvarium, including for mouse and human HSCs, indicating that endochondral ossification is dispensable for adult HSCNs. The model is currently being exploited to analyze leukemia/microenvironment interactions in live bone marrow. Conclusions: This study confirms and extends our recent statement considering the critical need for multimodal imaging (Lassailly F et al, Blood 2010). Further more, it demonstrates that combination of different imaging modalities for in-vivo and ex-vivo analysis is a powerful strategy allowing to shed a new light on the structure of the bone marrow microenvironment and improve our understanding of stem cells/niches interactions in health and disease. Disclosures: No relevant conflicts of interest to declare.

Erythropoietin couples erythropoiesis, B-lymphopoiesis, and bone homeostasis within the bone marrow microenvironment

Blood ◽  
2011 ◽  
Vol 117 (21) ◽  
pp. 5631-5642 ◽  
Author(s):  
Sofie Singbrant ◽  
Megan R. Russell ◽  
Tanja Jovic ◽  
Brian Liddicoat ◽  
David J. Izon ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Trabecular Bone ◽  
Bisphosphonate Therapy ◽  
Lineage Tracing ◽  
Bone Marrow Microenvironment ◽  
Bone Homeostasis ◽  
B Lymphopoiesis ◽  
Hematopoietic Stem ◽  
Erythroid Response

Abstract Erythropoietin (Epo) has been used in the treatment of anemia resulting from numerous etiologies, including renal disease and cancer. However, its effects are controversial and the expression pattern of the Epo receptor (Epo-R) is debated. Using in vivo lineage tracing, we document that within the hematopoietic and mesenchymal lineage, expression of Epo-R is essentially restricted to erythroid lineage cells. As expected, adult mice treated with a clinically relevant dose of Epo had expanded erythropoiesis because of amplification of committed erythroid precursors. Surprisingly, we also found that Epo induced a rapid 26% loss of the trabecular bone volume and impaired B-lymphopoiesis within the bone marrow microenvironment. Despite the loss of trabecular bone, hematopoietic stem cell populations were unaffected. Inhibition of the osteoclast activity with bisphosphonate therapy blocked the Epo-induced bone loss. Intriguingly, bisphosphonate treatment also reduced the magnitude of the erythroid response to Epo. These data demonstrate a previously unrecognized in vivo regulatory network coordinating erythropoiesis, B-lymphopoiesis, and skeletal homeostasis. Importantly, these findings may be relevant to the clinical application of Epo.

The Hypoxic Microenvironment in Acute Myelogenous Leukemia: Critical Role of CXCR4 in the Induction of HIF-1α.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1819-1819
Author(s):  
Michael Andreeff ◽  
Martin Dietrich ◽  
Paul Corn ◽  
Sergej Konoplev ◽  
Marina Konopleva ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Critical Role ◽  
Mapk Signaling ◽  
Bone Marrow Microenvironment ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Mrna Levels ◽  
Hematopoietic Stem ◽  
Hypoxic Conditions

Abstract The bone marrow microenvironment provides functional and structural support for both normal and leukemic hematopoietic stem cells. Importantly, the marrow microenvironment is known to be hypoxic. Given the many critical functions of HIF-1α, we investigated HIF-1α levels in leukemic cell lines and primary AML blasts. Surprisingly, HIF-1α expression was non-detectable in AML cells grown in suspension cultures under normoxic (21% O2) or hypoxic (1% O2) conditions. Normoxic co-cultures with bone marrow-derived mesenchymal stromal (MSC) or MS5 cells likewise did not induce HIF-1α, but hypoxic co-culture conditions induced HIF-1α protein without changes in HIF-1α mRNA levels, suggesting post-transcriptional regulation. Functionality of HIF-1α was confirmed by concomitant increase in the levels of glucose transporter glut-1, the HIF-1α downstream target. Inhibition of stroma-leukemia cell interactions with the small molecule CXCR4 inhibitor AMD3465 (Genzyme/Anormed) at 100 nM completely abrogated the induction of HIF-1α in HL-60 and MOLM13 AML cells. While SDF-1 was unable to induced HIF-1α under normoxic conditions, it did so under both physical (1% O2) and chemical (COCl2) hypoxic conditions, in two different cell lines. Inhibition of P13K (with LY294002) or MEK/ERK signaling (with CI-1040) abrogated HIF-1α induction under hypoxic conditions. Immunohistochemical staining of bone marrow samples from primary AML confirmed the presence of HIF-1α in leukemic cells localized adjacent to bone-lining stromal elements. Results suggest that the bone marrow microenvironment of AML is hypoxic in vivo; in leukemia cells HIF-1α induction under low oxygen tension depends on the presence of stromal cells; HIF-1α induction is dependent on SDF-1/CXCR4 and is mediated by activation of P13K and MAPK signaling. Altogether these findings suggest that SDF-1α/CXCR4 interactions contribute to the survival of leukemic cells via specific induction of HIF-1α signaling by the bone marrow microenvironment. Disruption of these interactions via CXCR4 inhibition strategies may suppress multiple pro-survival HIF-1α targets in leukemic cells.

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