Vascular Niche Research Articles

An Altered Bone Marrow Vascular Niche Impacts Normal Hematopoiesis and Tilts the Balance Between Myelopoiesis and Lymphopoiesis

The bone marrow niche is an important milieu where hematopoietic stem and progenitor cells (HSPCs) are maintained to ensure their lifelong contribution to hematopoiesis. Recent evidence has highlighted the critical importance of the perivascular bone marrow (BM) niche as the key host and regulator of HSPCs. Bone marrow endothelial cells (BMECs) are major components of the vascular niche. While studies have shown that an alteration in a component of the niche can affect hematopoiesis and promote the development of myeloproliferative disorders/myelodysplastic syndromes, it remains unclear how altered BMECs can impact hematopoiesis. To this end, we have generated a Tamoxifen (TAM)-inducible Tie2-CreER/LSL-KRasG12D;tdTomato mouse model to introduce an oncogenic KRas mutation specifically in adult endothelial cells. The tdTomato reporter overlaps with the CD31 and vascular endothelial growth factor receptor 2 (VEGFR2) endothelial cell markers and shows no detectable leakage into the adult hematopoietic compartment. To evaluate changes in hematopoiesis, we performed complete blood counts at 12 weeks post TAM injection and found that the Tie2-CreER/LSL-KRasG12D mice (KRasG12D mice) had significantly more leukocytes (p=0.031) and neutrophils (p=0.002) than controls. Flow cytometry analysis confirmed that the KRasG12D mice had a significantly higher percentage of myeloid cells with concurrent decrease in lymphocyte percentage in the peripheral blood (p=0.016). At 16 weeks post TAM injection, a significant decrease in B cells could also be noted in the blood of KRasG12D mice (p=0.028). Compared to controls, the KRasG12D mice displayed splenomegaly (p=0.025) and their spleens had a higher percentage of myeloid cells (p=0.002). There was an increase in the common myeloid progenitor compartment in the spleen and a significant increase in the granulocyte macrophage progenitor compartment (p=0.014) of KRasG12D mice. These mice also had an increase in the short-term hematopoietic stem cell (ST-HSC) compartment both in the BM and spleen. Colony forming assays revealed that KRasG12D mice had a higher number of total colonies formed from BM (p=0.044), spleen (p=0.007) and blood cells (p=0.56). Genotyping PCR showed no KRasG12D activation in hematopoietic cells, confirming that the observed phenotypes were due to an effect in BMECs. To complement our native inducible mouse model, we transplanted BM cells from syngeneic BoyJ mice into lethally irradiated Tie2-CreER;KRasG12D or KRasWT recipients. The endothelial KRasG12D recipientsdied between 75-200 days post transplantation (p=0.0079) while the KRasWT recipients remained alive. The KRasG12D recipients also displayed splenomegaly (p=0.004). Competitive transplant studies with donor cells from KRasG12D or KRasWT mice with competitor cells from syngeneic mice (CD45.1) showed that BM cells from the KRasG12D mice (CD45.2) outcompeted cells from KRasWT mice with a significantly higher percentage of CD45.2 donor chimerism in all blood lineages examined. To uncover any molecular events underpinning these hematopoietic changes, we performed quantitative real-time polymerase chain reaction. Our preliminary experiments from total BM RNA of KRasG12D or KRasWT mice indicate that there is a significant increase in VEGFα and a decrease in transforming growth factor β in KRasG12D mice, accompanying the above noted increase in the ST-HSC population. Collectively, our data provide strong evidence that an abnormal vascular niche caused by oncogenic insults in BMECs can disrupt normal hematopoiesis and promote a myeloproliferative phenotype, thereby implicating abnormal BMECs as novel contributors to blood pathogenesis. Studies are underway to further assess the molecular contributions from the disrupted vascular niche and the resulting HSPCs. Uncovering the mechanism of how altered BMECs can remodel hematopoiesis holds the exciting promise of better therapeutic strategies. DisclosuresNo relevant conflicts of interest to declare.

JAK2V617F-Mutant Vascular Niche Promotes JAK2V617F Clonal Expansion in Myeloproliferative Neoplasms

IntroductionThe myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by hematopoietic stem/progenitor cell (HSPC) expansion and overproduction of mature blood cells. The acquired mutation JAK2V617F plays a central role in these disorders. The mechanisms responsible for MPN HSPC expansion are not fully understood, limiting the effectiveness of current treatments. Endothelial cells (ECs) carrying the JAK2V617F mutation can be detected in patients with MPNs. In this work we test the hypothesis that the JAK2V617F-mutant vascular niche promotes JAK2V617F clonal expansion in MPNs.MethodsJAK2V617F Flip-Flop (FF1) mice (Radek Skoda, Switzerland) and Tie2-Cre mice (Mark Ginsberg, UC San Diego) were crossed to generate a strain in which human JAK2V617F is expressed specifically in hematopoietic cells and ECs (Tie2/FF1). Animal experiments were performed in accordance with the Institutional Animal Care and Use Committee guideline.ResultsPrimary lung ECs (CD45-CD31+) were isolated from Tie2/FF1 mice (JAK2V617F ECs) and age-matched littermate control mice (JAK2WT EC). JAK2V617F ECs proliferated to a greater extent than JAK2WT ECs. A vascular tube formation assay was performed as a measure of in vitro angiogenesis and revealed that JAK2V617F ECs had significantly increased angiogenesis compared to JAK2WT EC. (Figure 1)To examine the role of JAK2WT and JAK2V617F ECs in MPN hematopoiesis in vitro, marrow Lin-cKit+ HSPCs were isolated from Tie2/FF1 and age-matched littermate controls and cultured on a feeder layer of JAK2WT or JAK2V617F ECs under serum-free conditions. While there was no difference between JAK2V617F and JAK2WT HSPC proliferation when co-cultured with JAK2WT EC, the JAK2V617F HSPC displayed a relative growth advantage over the JAK2WT HSPC (1.24-fold, p = 0.041) when co-cultured on JAK2V617F EC.Next, the effects of JAK2V617F-bearing vascular niche on MPN hematopoiesis were studied in vivo using marrow transplantation assays. First, wild-type CD45.1 marrow cells were transplanted into lethally irradiated (950cGy) 8-14 week old Tie2/FF1 mice or controls (CD45.2) (n=7 in each group). During a >8-month follow up, all wild-type recipients displayed full donor engraftment, while 4 of 7 Tie2/FF1 recipient mice displayed recovery of JAK2V617F-mutant hematopoiesis (mixed donor/recipient chimerism), suggesting that the JAK2V617F-mutant vascular niche may protect the JAK2V617F-mutant HSPCs from the lethal irradiation. Although marrow and splenic hematopoietic progenitors were significantly increased in the Tie2/FF1 recipients, there was no difference in peripheral blood (PB) cell count or PB hematopoietic progenitor cell numbers between the Tie2/FF1 mice and control mice. (Figure 2)Second, we performed a competitive marrow transplantation experiment in which 5 x 105 CD45.2 donor marrow cells from Tie2/FF1 mice were injected intravenously together with 5 x 105 competitor CD45.1 wild-type marrow cells intro lethally irradiated Tie2/FF1 mice or control mice (CD45.2) (n=5 in each group). During a 4-month follow up, Tie2/FF1 recipients displayed lower PB CD45.1 (i.e. wild-type donor) chimerism than the control mice (p = 0.025), suggesting that the mutant vascular niche in Tie2/FF1 promoted the JAK2V617F clonal maintenance/expansion and/or inhibited the wild-type hematopoiesis. By 18 weeks post-transplant, Tie2/FF1 recipients developed a MPN phenotype with elevated WBC count (22.7 vs. 9.85 x 109/L, p = 0.049), platelet count (5336 vs. 1029 x 109/L, p = 0.033), increased splenomegaly, increased total marrow and splenic hematopoietic progenitors, and increased CD150+CD48- E-SLAM cells (a highly enriched HSPC population) in both marrow (5-fold, p = 0.0004) and spleen (5.5-fold, p = 0.000004) compared to the control mice. To begin to understand the EC signals responsible for JAK2V617F clonal expansion and MPN pathogenesis, qRT-PCR of purified marrow ECs identified up-regulation of MPL and CXCL12 in both arterial (CD45-CD31+Sca1+) and sinusoidal (CD45-CD31+Sca1-) marrow ECs in Tie2/FF1 mice compared to controls.ConclusionsOur study establishes that JAK2V617F-bearing ECs form an important part of the MPN HSPC niche and contribute to mutant stem/progenitor cell expansion both in vitro and in vivo. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Ifnα Mediated Remodeling of the Bone Marrow Stem Cell Vascular Niche

In the bone marrow (BM), endothelial cells (ECs) are a major component of the hematopoietic stem cell (HSC) vascular niche and are a first line of defense against inflammatory stress and infection. The primary response of an organism to infection involves the synthesis of immune-modulatory cytokines, including interferon alpha (IFNα). In the BM, IFNα induces rapid cell cycle entry of HSCs in vivo. However, the effect of acute IFNα treatment on BM ECs has not been described. Here, we demonstrate that IFNα leads to rapid stimulation of BM ECs in vivo, resulting in increased BM vascularity and vascular leakage. We find that stimulation of BM ECs involves the expression of key inflammatory and EC-stimulatory markers, including ESAM, VE-Cadherin and Laminin. Using the anti-VEGF antibody, Avastin, we could confirm that IFNα-mediated activation of BM ECs is dependent in part on VEGF signaling in BM hematopoietic cell types, including HSCs. Thus, this implies a role for HSCs in remodeling of the BM niche in vivo followinginflammatory stress. Taken together, these data increase our current understanding of the relationship between HSCs and the BM niche under inflammatory stress. In addition, we have clarified the response of BM niche ECs to acute IFNα treatment in vivo. IFNα treatment has been associated with anti-angiogenic effects in patients and therefore our described acute response of the BM vasculature may additionally inform future regimes of combination therapy. DisclosuresNo relevant conflicts of interest to declare.

Vascular E-Selectin Protects Leukemia Cells from Chemotherapy By Directly Activating Pro-Survival NF-Kb Signalling - Therapeutic Blockade of E-Selectin Dampens NF-Kb Activation

The vascular adhesion molecule E-selectin is a key component of the Bone Marrow (BM) Haematopoietic Stem Cell (HSC) niche prompting HSC to proliferate at the expense of self-renewal (Winkler, Nat Med 2012).Only 3 - 5% of BM endothelial cells express E-selectin in steady state however E-selectin is greatly upregulated (5 - 10 fold) in BM of mice with acute myeloid leukemia (AML) raising the question; how do AML stem cells (LSC) respond to E-selectin at the vascular niche & does E-selectin signalling in AML & HSC differ?Using models of murine AML generated by retroviral transduction of MLL-AF9 or AML1-ETO oncogenes, we found leukemic blasts rapidly upregulate E-selectin-binding upon oncogenic transformation. Furthermore E-selectin adhesion promoted LSC survival to cytarabine in vitro as well as in vivo. LSC survival to chemotherapy in wildtype compared to E-selectin knockout (E-/-) mice quantified by rigorous limiting-dilution transplantation assay of 1%, 0.1%, 0.01% femur BM demonstrated that E-selectin deletion increased sensitivity of LSC to high-dose cytarabine therapy ~11-fold (900mg/kg cytarabine n=6 donors &15 recipients/gp p=0.0037). Thus E-selectin is a critical vascular niche component mediating LSC chemo resistance. Importantly these findings could be replicated by administration of a potent small molecule glycomimetic E-selectin antagonist (GMI-1271) to wt mice. Furthermore treatment with GMI-1271 (40mg/kg bidaily) for 10 days in combination with standard mouse version of 7+3 induction chemotherapy (5 days cytarabine 100mg/kg; 3 days doxorubicin 1mg/kg) was able to significantly double mouse survival over chemotherapy alone (p=0.0054; no chemotherapy median survival 25 d, AraC/Dox alone 32 d, AraC/Dox plus GMI-1271 survival 41 d; n=8 mice/gp).To understand mechanisms of this chemo-sensitisation, mice with MLL-AF9 monomyelocytic (11q23 translocation) or AML1-ETO granulocytic t(8;21) -induced AML were administered GMI-1271 or vehicle control for 5 days before sorting BM AML blasts for RNA sequencing. Analysis of differentially expressed transcripts by CuffDiff / DSeq2 revealed 170 RNAs differed following in vivo E-selectin blockade. KEGG pathway analysis indicated a pathway potentially dampened in AML blasts following GMI-1271 administration was PI3K - NF-kB signalling - raising the hypothesis that adhesion to E-selectin activates pro-survival NF-kB signalling in AML cells leading to enhanced chemoresistance.Using two in vitro assays, we confirmed E-selectin to be unique among vascular adhesion molecules tested in being able to directly activate NF-kB. Activation of NF-kB was only observed upon E-selectin mediated adhesion & was not observed following adhesion to P-selectin, PECAM-1 or VCAM-1 using either myeloid NF-kB GFP reporter cell lines or by induction of p65 NF-kB (Ser 536). Importantly E-selectin mediated NF-kB activation was completely inhibited when E-selectin antagonist GMI-1271 added.Assays repeated in presence of a specific NF-kB activation antagonist (BMS-345541 10uM 24hrs) demonstrated that NF-kB blockade alone reversed E-selectin-mediated chemoresistance in vitro. Upstream blockade of E-selectin by GMI-1271 not only inhibits NF-kB activation but also mobilizes LSC out of the protective BM niche & prevents re-entry thereby breaking the chemo resistance observed with these cells. A Phase I/II Clinical trial to study efficacy of GMI-1271 in combination with chemotherapy in AML patients (NCT02306291) is currently in progress DisclosuresWinkler:GlycoMimetics: Research Funding. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Levesque:GlycoMimetics: Equity Ownership.

Hypoxia Inducible microRNA-210 Regulates the DIMT1-IRF4 Oncogenetic Axis in Multiple Myeloma

Background: It is known that gene expression patterns in tumor cells are heterogeneous even in the same individual, not only in solid tumors but also in hematological malignancies, including multiple myeloma (MM). MM is characterized by the accumulation of a population of malignant plasma cells within the bone marrow (BM) and microenvironment (endosteal and vascular niches). Hypoxic oxygen stress occurs in the microenvironment, leading to various epigenetic gene alterations. For instance, hypoxia lead to cell cycle arrest, glycolysis, epithelial-mesenchymal transition (EMT)-related machinery, drug resistance and immature phenotype decreasing expression of CD138 and IRF4. Recently, it is reported that a microRNA (miRNA) in exosomes of myeloma treated in hypoxia induces vessel formation and promotes survival of myeloma cells via upregulation of HIF-1. However, to the best of our knowledge, there have been no reports describing expression changes of miRNAs in myeloma cells during hypoxia. The aim of this study was to detect epigenetically regulated miRNA and essential targets that could be affected by hypoxia in MM.Methods: To identify hypoxia-induced miRNA(s) and their target gene(s), we conducted whole miRNA and mRNA microarray screening using myeloma cell lines (MM.1S, RPMI-8226, KMS12BM, KMS11, and U266) and primary samples (n=4) that were subjected to chronic hypoxia conditions (1% O2 for 48 hr). We purified CD38++ cells (CD38: a marker for activated B-cells and plasma cells) from primary samples, and then conducted functional analysis of target gene/proteins of identified hypoxia inducible miRNA.Results: We recognized a significant upregulation of microRNA-210 (miR-210) under chronic hypoxia. We explored target genes of miR-210 as well as downregulated genes by hypoxia comprehensively, and we found that five candidate genes including DIMT1, CIAPIN1, TTC13, ARMC1, and NOL12 by TargetScan program. Among these, 18S rRNA base methyltransferase DIMT1, which is necessary for the production of ribosome, was the most likely candidate target of miR-210 in myeloma cells because the product was only directly reduced by miR-210 transduction of myeloma cells. We confirmed significant upregulation of miR-210 and downregulation of DIMT1 in primary samples (n=15) cultured in hypoxia by q-RT-PCR. In the examination of the myeloma patient samples on several GEO data sets, we found that expression of DIMT1 increased significantly with disease progression, and that DIMT1 had a positive correlation of IRF4. Notably, immunohistochemical analysis revealed that DIMT1 was strongly stained at myeloma cells but not other BM cells including various lymphocytes. The knock down of DIMT1 for MM.1S, H929, and KMS11 cell lines lead to apoptosis and downregulation of IRF4. Interestingly, miR-210 transduction also reduces expression of IRF4. Furthermore, we found that pan-HDAC inhibitors (panobinostat and vorinostat) inhibit cell survival via downregulation of the DIMT1-IRF4 axis in myeloma cells.Conclusion: By screening for targets of hypoxia inducible miR-210, we identified DIMT1 as a novel diagnostic marker and therapeutic target for all molecular subtypes of MM. Our results suggest that the master antiapoptosis regulator might be different between normoxia and hypoxia. In hypoxia, HIFs and/or the HIF-miR-210 axis may convert myeloma cells to an antiapoptotic phenotype. However, in normoxia, DIMT1 was highly expressed in myeloma cells and, herein, activation might convert myeloma cells to the antiapoptotic phenotype. [Display omitted] DisclosuresIkeda:Novartis: Research Funding; Pfizer: Research Funding; Daiichi Sankyo: Research Funding; Kyowa Kirin: Research Funding; Otsuka: Research Funding; Toyama Chemical: Research Funding; Chugai: Research Funding; AsahiKasei: Research Funding; Eisai: Research Funding; Fujimoto: Research Funding. Kitadate:Daiichi Sankyo: Research Funding; Kyowa Kirin: Research Funding; Otsuka: Research Funding; Toyama Chemical: Research Funding; Chugai: Research Funding; AsahiKasei: Research Funding; Eisai: Research Funding; Fujimoto: Research Funding; Pfizer: Research Funding; Novartis: Research Funding. Abe:Novartis: Research Funding; Pfizer: Research Funding; Daiichi Sankyo: Research Funding; Kyowa Kirin: Research Funding; Otsuka: Research Funding; Toyama Chemical: Research Funding; Chugai: Research Funding; AsahiKasei: Research Funding; Eisai: Research Funding; Fujimoto: Research Funding. Takahashi:Pfizer: Honoraria, Research Funding; BMS: Honoraria; Novartis: Honoraria, Research Funding. Tagawa:Kyowa Kirin: Research Funding; Otsuka: Research Funding; Chugai: Research Funding; AsahiKasei: Research Funding; Eisai: Research Funding; Toyama Chemical: Research Funding; Fujimoto: Research Funding; Daiichi Sankyo: Research Funding; Pfizer: Research Funding; Novartis: Research Funding.

Transforming Growth Factor- β1 Signaling in Vascular Endothelial Cells Inhibits Hematopoietic Stem Cell Regeneration

Endothelial cells (EC) are known to be essential for hematopoietic regeneration; however, little is known about the pathways that regulate this activity. By modeling endothelial-dependent HSC interactions in vitro, we found that human umbilical vein endothelial cells (HUVEC) had a markedly reduced capacity to regenerate functional CD150+LSK cells (HSC) compared to other sources of arterial and venous EC. Transcriptional profiling revealed the overexpression of transforming growth factor- β1 (TGF-β1) in HUVEC and indicated that TGF-β1 driven transcriptional programs are highly active in these cells, a finding consistent with autocrine TGF-β1 signaling. Functional studies demonstrated that HSC regeneration by EC was potently inhibited by TGF-β1 and augmented by the ALK5 inhibitor SB431542, in a dose-dependent manner. Importantly, exposure of EC alone to TGF- β1 was sufficient to attenuate subsequent HSC self-renewal. Transcriptome analysis also identified hepatocyte growth factor (HGF) as a candidate EC-derived factor with the potential to enhance hematopoietic regeneration. HGF treatment of HUVEC activated endothelial Akt signaling and led to a >10-fold increase in HSC regeneration that could be blocked by the c-Met inhibitor PF04217903. HGF treatment also dramatically increased long-term multi-lineage hematopoiesis from HUVEC regenerated HSC. Our findings reveal a novel suppressive role for TGF-β1 in the vascular niche and demonstrate that EC-derived growth factors such as HGF have the potential to attenuate this suppression and significantly enhance HSC regeneration. DisclosuresNo relevant conflicts of interest to declare.

The Pro-Tumorigenic Vascular Niche Sustains the T-Cell Acute Lymphoblastic Leukemia Phenotype and Fosters Resistance to Therapy

Introduction. T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous malignancy associated with a high risk of treatment failure. Efforts to improve outcomes have focused on underlying genetic defects. However, new evidence suggests that the microenvironment can foster drug resistance/relapses. Identification of factors that contribute to microenvironment-mediated chemo-refractoriness remains an important challenge. Here, we sought to construct an in vitro platform to dissect tumor-host interactions and to optimize drug treatments using Patient-Derived Tumor Xenograft models (PDTX) of high risk adult T-ALL and engineered human endothelial cells.Methods. T-ALL PDTX were established and serially passaged in NSG mice. Engraftment was monitored by flow cytometry of peripheral blood and/or MRI. Mice were sacrificed and leukemic cells were harvested from the spleen/bone marrow. To determine the ex vivo growing conditions, we first cultured a panel of 8 "bona fide" T-ALL cell lines and 11 PDTX cells alone in complete RPMI 20% FCS supplemented with IL2, IL12, IL15 and IL7; or co-cultured with human E4-ORF1 endothelial cells (ECs) without ILs in complete RPMI 20% FCS or serum/cytokine-free media. CDK4/6, MEK, PI3K and JAK inhibitors were used at 0.1 and 1 µM alone and in combination. Cell titer glo, cell titer blue, Annexin-V and S-cell cycle analysis were used as readouts. Total RNA from cells before and after co-culture was extracted for paired-end RNA sequencing on an Illumina HiSeq2500.Results. To study the supporting role of ECs, we first co-cultured ECs with T-ALL cell lines in vitro (serum/cytokine free co-culture) and showed that ECs could reproducibly sustain the viability of 3/8 cell lines (Loucy, KOPTK1, P12 Ichikawa) serum/cytokine-free media. A partial rescue was seen with 3 additional lines (HPB-ALL, CCRF-CEM, CUTLL1), while 2 (KE37, DND41) underwent massive cell death. We next tested whether either ILs or CXCL12 could provide anti-apoptotic signals and demonstrated that KOPTK1 and Loucy were only partially rescued by IL15 or CXCL12. Conversely, IL7, although capable of inducing a robust upregulation of pSTAT5, had no effect (CCRF-CEM and CUTLL1). We then characterized 11 PDTX from 15 high-risk adult T-ALL patients. All PDTX were serially propagated and caused T-ALL in subsequent NSG mice (massive spleen and bone marrow infiltration with extensive paravertebral mass associated with paralysis and multi-organ involvement). Genomic analysis (RNA-seq) demonstrated a high concordance between primary (pre-implant) and PDTX samples. All of them were extensively studied ex vivo, demonstratingthat T-ALL PDTX cells could only survive in ILs supplemented media, even better if enriched of growth factors and supplements for the expansion of human hematopoietic cells. However, when PDTX cells were treated with targeting compounds they all underwent massive apoptosis. Conversely, individual PDTX T-ALL could be selectively rescued by ECs, allowing the construction of individual drug response profile. To extend these data, 7 PDX T-ALL samples were screened against a 430-targeted compound library in supplemented RPMI or Stem Span media. Results indicated differential cell killing and gain (NFKB, BTK) and loss (TP-53, IGF-1R) of targets.Conclusions. These data clearly demonstrate a key role of aberrantly activated vascular niche in T-ALL cell maintenance and drug resistance. We envisage that drug screening of EC+T-ALL will lead to the identification of actionable targets in each individual patient. Our report supports the potential for future personalized curative strategies aimed at targeting both tumor cells and host tissue supporting niche elements disrupting pro-tumorigenic signals within leukemia cell niches. DisclosuresFoà:Roche: Consultancy, Speakers Bureau; Genentech: Consultancy; Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; BMS: Consultancy; Pfizer: Speakers Bureau; Ariad: Speakers Bureau. Rafii:Angiocrine Bioscience: Equity Ownership, Other: Non-paid consultant.

Increased Vascular Permeability in the Bone Marrow Microenvironment Contributes to Acute Myeloid Leukemia Progression and Drug Response

The biological and clinical behavior of hematological malignancies are not only determined by the properties of the leukemic cells themselves, but are also highly affected by interaction with the microenvironment, pointing to the existence of an active crosstalk between the two compartments. Previous studies showed that acute myeloid leukemia (AML) cells actively modify endothelial cells ex vivovia several pathways, mainly mediated by VEGF. However, as anti-VEGF therapies haven't produced successful results in clinical trials, an extensive study of the crosstalk between AML and the vascular niche in the bone marrow (BM) is required to provide new therapeutic strategies. In the present study we combined the use of mouse models of AML, human AML patient-derived xenografts (PDX) and direct analysis on patient-derived BM biopsies to provide a global, reliable picture of the bone marrow vasculature in AML disease. We found several abnormalities in the vascular architecture and function in PDX, such as increased number of endothelial cells, increased microvascular density (MVD), decreased vascular mean diameter and increased hypoxia. Furthermore, using two-photon confocal intravital imaging we witnessed increased vascular permeability upon AML engraftment, observed homogeneously among different PDX. Interestingly, induction chemotherapy failed to normalize the vascular permeability in the BM, despite significant reduction in AML engraftment. We identified increased nitric oxide (NO) as a major mediator of the AML-induced vascular leakiness in the BM. Increased levels of NO and activated NOS3 were found in PDX and in an independent cohort of patient-derived BM biopsies. Strikingly, inhibition of NO production using genetic and pharmacological approaches reduced the vascular permeability, potentiated the normal HSC function and significantly improved treatment response in PDX. These results strongly support the notion of a primary function of the vascular permeability in AML progression, drug response and in affecting normal stem cell function, and they call for clinical trials incorporating NOS inhibitors during the remission phase to target the abnormal vascular niche and improve the treatment response. DisclosuresNo relevant conflicts of interest to declare.

Interferon-Gamma Impairs Expansion and Hematopoietic Support of Bone Marrow Mesenchymal Stromal Cells

Maintenance of hematopoietic stem cells (HSCs) and regulation of their quiescence and self-renewal is critical for maintaining a lifelong supply of blood cells. The ability of HSCs to stay quiescent is thought to depend on their specific niche in the bone marrow (BM). Mesenchymal stromal cells (MSC) in the BM are multipotent stem cells that form part of the vascular HSC niche and provide micro-environmental support to HSCs both in vivo and upon expansion ex vivo. Culture-expanded MSCs also exhibit immunomodulatory properties that can be enhanced by pre-treatment with interferon-gamma (IFN-γ). BM MSC are thus attractive candidates for cellular therapy after hematopoietic stem cell transplantation, for promoting rapid hematopoietic recovery and reducing the incidence or severity of graft versus host disease. Although IFN-γ pre-treatment can improve the immunomodulatory properties of MSCs, elevated IFN-γ levels have also been associated with anemia and BM failure in multiple chronic inflammatory diseases. While the impact of IFN-γ on HSC has been elucidated in recent years, it remains largely unknown whether IFN-γ can also influence hematopoietic support by BM stromal cells.In this study, we aim to elucidate the impact of IFN-γ on hematopoietic support of BM MSC.We show that in vitro expansion of primary BM MSC cultures from healthy donors was significantly reduced in the presence of IFN-γ, and this effect could be reproduced in the BM stromal cell line MS-5. Concurrently, IFN-γ diminished the clonal capacity of BM MSC, as measured by CFU-F assays. In addition, BM MSC that were pre-stimulated with IFN-γ produced significantly lower levels of CXCL12, suggesting a loss of hematopoietic support potential. Indeed, support of CD34+ hematopoietic stem and progenitor cells (HSPC) in a co-culture assay was greatly reduced in when MSC were pre-treated with IFN-γ.To determine the impact of IFN-γ on BM MSC in vivo, we investigated the BM stromal compartment of IFN-γ AU-rich element deleted (ARE-Del) mice, which constitutively express IFN-γ in steady state conditions. FACS analysis revealed a remodeling of the BM stromal compartment in ARE-Del mice compared to littermate controls, with significantly fewer MSCs, identified as CD45-Ter119-CD31-CD51+PDGFRa+ cells. Numbers of other stromal cell subsets, such as osteoblasts and fibroblasts, were not altered.The reduction of BM MSC in ARE-Del mice coincided with a loss of quiescence in HSCs; only 35% of long term HSC (LT-HSC) in ARE-Del mice were quiescent, compared to 70% in WT mice, as determined by Ki-67 staining. Loss of quiescence in LT-HSC did not lead to increased self-renewal, but rather induced increased differentiation towards short-term HSC and multi-potent progenitors.We then sorted LT-HSC from WT and ARE-Del mice and performed in vitro HSC culture assays in the absence of IFN-γ. Absolute numbers of LT-HSC were rapidly decreased in ARE-Del compared to WT cultures after 3 and 7 days of HSC culture, while numbers of more differentiated progenitors were increased. These data indicate that an IFN-γ-mediated loss of BM MSC in ARE-Del mice leads to loss of quiescent LT-HSCs and induces a tendency towards HSC differentiation over self-renewal.In conclusion, we have shown that IFN-γ has a negative impact on expansion and hematopoietic support of BM MSC in vitro and in vivo across species. Although IFN-γ treatment enhances the immunomodulatory function of MSCs in a clinical setting, it is obvious from our data that IFN-γ impairs their HSC supporting function. These data also provide more insight in the underlying mechanism by which IFN-γ contributes to the pathogenesis of anemia and BM failure. DisclosuresNo relevant conflicts of interest to declare.

Loss of Adrenergic Nerves in the Bone Marrow Microenvironment Drives an Aging HSC Niche Phenotype

Hematopoietic stem cell (HSC) aging is accompanied by an expansion of myeloid-biased HSCs with declined long-term self-renewal functions, contributing to the development of hematologic malignancies. Recent studies have suggested a role for the bone marrow microenvironment in HSC aging but the extrinsic molecular mechanisms driving their aging remain largely unknown. Mesenchymal stem cells (MSCs) form a critical component of the HSC niche, promoting HSC quiescence and balanced differentiation. Some MSC activity is found associated with arterioles, which are densely innervated by the sympathetic nervous system (SNS). We used tridimensional whole-mount bone marrow confocal immunofluorescence imaging of HSCs and vascular structures to investigate the effects of aging on the HSC microenvironment. Comparative bone marrow analyses of aged (20-24 months) C57BL/6 and Nestin-GFP transgenic mice (where GFP marks MSCs) with young (2 month-old) C57BL/6 mice, revealed significant reductions in sympathetic innervation (3-fold, p<0.01) as determined by staining for tyrosine hydroxylase expressing nerve fibers. The SNS neuropathy of aged mice was accompanied with vascular remodeling characterized by an increase in total vascular density marked by CD31+/CD144+ (2-fold, p<0.01) and shortening of arterioles marked by CD31+/CD144+ and Nestin-GFP+ (5-fold reduction, p<0.001), reduction in α-smooth muscle actin (αSMA) pericytes (12-fold, p<0.01) and expansion of Nestin+ MSCs (4 fold, p<0.05). Whereas a subset of HSCs (~35%) are associated with arterioles in young mice, analyses of HSC localization in aged bone marrow revealed that HSCs were redistributed and expanded away from arterioles (p=0.0047). Functional analysis revealed a diminished in vitro clonal capacity of aged MSCs (determined by CFU-F and mesensphere assays; 3-fold, p<0.0001), reduced HSC maintenance activity determined by the ability to produce HSC maintenance factors (Cxcl12, Scf and Angpt1, 2-fold, p<0.01), and increased proliferative state (2-fold, p<0.0001), all indicative of reduced activity. Since SNS nerves regulate circadian egress of HSCs through regulation of MSC activity, we evaluated the circadian oscillations of HSCs in blood and niche factors in Nestin-GFP+ stromal cells. Aged mice exhibited ablated oscillations of progenitor release determined by peripheral blood colony-forming units in culture (CFU-C/ml blood) and phenotypic progenitors (LSK/ml blood). Furthermore, unlike the robust circadian oscillations of Cxcl12 levels of young MSCs, no circadian expression pattern was detected in aged mice, suggesting that the SNS neuropathy associated with aging has functional consequences by leading to disrupted circadian rhythms and expanded MSC populations with reduced HSC maintenance activity. To determine whether the loss of SNS nerves drives bone marrow vascular remodeling and HSC aging, we surgically sympathectomized young Nestin-GFP mice by unilateral microsection of both the sciatic and femoral nerves, while the sham-operated contralateral side served as control. Strikingly, four months following surgery, phenotypic HSCs selectively expanded in sympathectomized femurs (2-fold, p<0.05) and exhibited myeloid-biased differentiation upon competitive bone marrow transplantation compared to control (3-fold, p<0.01). Furthermore, similar to our observations in aged mice, chronic surgical sympathectomy (16 weeks) induced dramatic "aging-like" alterations of bone marrow arteriolar structures characterized with arteriolar shortening and loss of αSMA+ and Nestin-GFP+ arterioles (4-fold, p<0.01; 3 fold, p<0.001). These results thus suggest that aging-associated sympathetic neuropathy and the loss of β-adrenergic signals in the bone marrow microenvironment drive aging of HSCs by remodeling vascular niches and thus represent a potent therapeutic target for stem cell rejuvenation. DisclosuresFrenette:Pfizer: Consultancy; GSK: Research Funding; PHD Biosciences: Research Funding.

Alteration of the Hematopoietic Components, Mesenchymal Stromal Cells and Vascular Elements in the Bone Marrow Niche in Myelodysplastic Syndrome: Refractory Cytopenia with Multilineage Dysplasia

Introduction:The myelodysplastic syndromes (MDS) are clonal disorders characterized by cytopenias and abnormal hematopoiesis. Though there are reports of perturbations in the hematopoietic stem cells (HSC) and the mesenchymal stromal cells (MSC) as well as other elements of the bone marrow (BM) niche in an instructive or permissive manner leading to the genesis of ineffective hematopoiesis in this condition, most studies have evaluated single elements. Here we demonstrate altered HSC, MSC and the vascular niche elements in patients with MDS - Refractory cytopenia with multilineage dysplasia (RCMD).Methods and results: Bone marrow aspirates from patients with RCMD (n=12, mean age 54.33±14.51 years) were compared with those from age-matched controls (n=18, mean age 47.78±18.60 years) who had a ’normal’ marrow obtained for other diagnostic purposes. Cytogenetic analysis showed abnormal karyotypes in seven patients and normal karyotypes in five. The abnormalities seen were as follows: deletion 7q/monosomy 7 (four patients), deletion 5q (three patients), loss of Y (two patients), monosomy 5, deletion 20q (one each), complex karyotypes (four patients). Phenotypic enumeration of HSPCs revealed a marked decrease in the frequency of highly purified HSCs (Lin-CD34+CD38-CD90+CD45RA-) in RCMD (0.04135±0.01748%, n=11) when compared to controls (0.3168±0.05266%, n=13, p<0.0001) (Figure 1). Patients with RCMD also had increased common myeloid progenitors (CMP) (3.221 ± 0.7478%, n=8) compared to control (1.243±0.4463%, n=10, p<0.0302) and loss of granulocyte-macrophage progenitors (GMP) (0.4863±0.1638%, n=8) compared to controls (2.047±0.5422%, n=10, p<0.0242) (Figure 2).Assessment of the frequency of de novo MSCs (CD31-CD45/CD71- population) expressing CD271 and/or CD146, indicating the more primitive population, in total nucleated cells showed increased CD271+CD146-MSCs (0.632±0.2, n=5) compared to controls (0.2000±0.05158, n=6, p<0.0401) as also the CD271+CD146+ MSCs (0.2900±0.09803, n=5) in RCMD patients when compared to controls (0.02861±0.01354, n=6, p<0.0172) (Figure 3). We also evaluated in vitro cultured MSCs (P4) in these patients. CD271+CD146+ MSCs within total cultured MSCs were higher (0.1900±0.06429%, n=3) than in controls (0.0040±0.0040%, n=3, p<0.0447). RCMD MSCs had significantly lower proliferation index (32±3.7%, n=3) compared to controls (60±9.2%, n=3, p<0.0479). Cell cycle analysis of MSC showed significantly lower numbers in G0 in RCMD (0.1567 ± 0.07881 %, n=3) compared to control (0.6400 ± 0.1007 %, n=3; p<0.0194). Apoptosis was much higher in RCMD MSCs (2.700±0.8007%, n=3) compared to controls (0.03633±0.01802%, n=3, P<0.0292). No significant differences in expression profile of stem cell maintenance related cytokines and growth factors (CXCL12a, SCF, VEGF, ANGPT and LIF ) or components of Notch (Notch1, Notch3, Jagged-1, Delta like-1 and Hes1) and Wnt (Dkk1 and Dkk-2) pathways were found between RCMD and control MSCs within the limited numbers evaluated. Interestingly, unlike controls, immunofluorescence imaging of bone marrow trephine from MDS-RCMD revealed CD271+CD146+ MSCs co-localized with sinusoids and in direct contact with CD34+ (<5% blast) HSC/progenitor cells (HSPCs).Conclusion: Our data shows that in patients with MDS-RCMD, both qualitative and quantitative abnormalities exist in the HSC, HSPC and niche elements. We also show that the cytopenia could be related to decreased numbers of primitive HSCs and a differentiation arrest at the CMP stage. Primitive MSCs are reduced, and those that exist show poorer proliferative and survival features. Further studies are needed to understand the cause and effect relationship of these changes. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Endothelial-specific inhibition of NF-κB enhances functional haematopoiesis

Haematopoietic stem cells (HSCs) reside in distinct niches within the bone marrow (BM) microenvironment, comprised of endothelial cells (ECs) and tightly associated perivascular constituents that regulate haematopoiesis through the expression of paracrine factors. Here we report that the canonical NF-κB pathway in the BM vascular niche is a critical signalling axis that regulates HSC function at steady state and following myelosuppressive insult, in which inhibition of EC NF-κB promotes improved HSC function and pan-haematopoietic recovery. Mice expressing an endothelial-specific dominant negative IκBα cassette under the Tie2 promoter display a marked increase in HSC activity and self-renewal, while promoting the accelerated recovery of haematopoiesis following myelosuppression, in part through protection of the BM microenvironment following radiation and chemotherapeutic-induced insult. Moreover, transplantation of NF-κB-inhibited BM ECs enhanced haematopoietic recovery and protected mice from pancytopenia-induced death. These findings pave the way for development of niche-specific cellular approaches for the treatment of haematological disorders requiring myelosuppressive regimens.

Relevance of epithelial-to-pericyte transition in cancer

ABSTRACTThe relevance of epithelial-to-mesenchymal transition (EMT) in cancer is still under debate. Recently, we reported that EMT bestows key pericyte properties on cancer cells and may thus represent epithelial-to-pericyte transition (EPT). Carcinoma cells undergo EPT to stabilize blood vessels and fuel primary tumor growth. Association of EPT cancer cells with vascular niches may also promote resistance to therapy.

Periosteum tissue engineering in an orthotopic invivo platform.

The periosteum plays a critical role in bone homeostasis and regeneration. It contains a vascular component that provides vital blood supply to the cortical bone and an osteogenic niche that acts as a source of bone-forming cells. Periosteal grafts have shown promise in the regeneration of critical size defects, however their limited availability restricts their widespread clinical application. Only a small number of tissue-engineered periosteum constructs (TEPCs) have been reported in the literature. A current challenge in the development of appropriate TEPCs is a lack of pre-clinical models in which they can reliably be evaluated. In this study, we present a novel periosteum tissue engineering concept utilizing a multiphasic scaffold design in combination with different human cell types for periosteal regeneration in an orthotopic invivo platform. Human endothelial and bone marrow mesenchymal stem cells (BM-MSCs) were used to mirror both the vascular and osteogenic niche respectively. Immunohistochemistry showed that the BM-MSCs maintained their undifferentiated phenotype. The human endothelial cells developed into mature vessels and connected to host vasculature. The addition of an invitro engineered endothelial network increased vascularization in comparison to cell-free constructs. Altogether, the results showed that the human TEPC (hTEPC) successfully recapitulated the osteogenic and vascular niche of native periosteum, and that the presented orthotopic xenograft model provides a suitable invivo environment for evaluating scaffold-based tissue engineering concepts exploiting human cells.

JAK2V617F-mutant vascular niche contributes to JAK2V617F clonal expansion in myeloproliferative neoplasms

The myeloproliferative neoplasms (MPNs) are characterized by hematopoietic stem/progenitor cell (HSPC) expansion and overproduction of blood cells. The acquired mutation JAK2V617F plays a central role in these disorders. Mechanisms responsible for MPN HSPC expansion is not fully understood, limiting the effectiveness of current treatments. Endothelial cells (ECs) carrying the JAK2V617F mutation can be detected in patients with MPNs, suggesting that ECs are involved in the pathogenesis of MPNs. Here we report that JAK2V617F-bearing primary murine ECs have increased cell proliferation and angiogenesis in vitro compared to JAK2WT ECs. While there was no difference between JAK2V617F and JAK2WT HSPC proliferation when co-cultured with JAK2WT EC, the JAK2V617F HSPC displayed a relative growth advantage over the JAK2WT HSPC when co-cultured on JAK2V617F EC. In addition, the thrombopoietin (TPO) receptor MPL is up regulated in JAK2V617F ECs and contributes to the maintenance/expansion of the JAK2V617F clone over JAK2WT clone in vitro. Considering that ECs are an essential component of the hematopoietic niche and most HSPCs reside in the perivascular niche, our studies suggest that the JAK2V617F-bearing ECs form an important component of the MPN vascular niche and contribute to mutant stem/progenitor cell expansion, likely through a critical role of the TPO/MPL signaling axis.

Abstract B035: CXCL8/CXCR1 signaling promotes angiogenesis and hematopoietic stem and progenitor cell function

Abstract CXCL8 (IL-8) is a chemokine with pleiotropic roles in host defense, angiogenesis and tumor metastasis. CXCL8 and its specific receptor, CXCR1, are broadly expressed within the hematopoietic and vascular systems. In an effort to identify novel secreted factors with effects on hematopoietic stem and progenitor cell (HSPC) function, we have recently identified CXCL8/CXCR1 signaling as a positive regulator of HSPC colonization of the zebrafish caudal hematopoietic territory (CHT). The CHT is a vascular niche that serves as the primary site of hematopoiesis from 36 hours post fertilization (hpf) to 6 days post fertilization (dpf). This observation raised the question whether CXCR1 signaling might induce dynamic changes in the CHT that favor HSPC colonization. CXCR1 was expressed at high levels in endothelial cells using a kdrl(VEGFR2):CXCR1;kdrl:mCherry double transgenic line. The CHT was imaged by fluorescence confocal microscopy, reconstructed in 3 dimensions and the volume measured using digital image analysis software. Overexpression of CXCR1 within the endothelial cells of these animals increased the volume of the CHT by 28% (p = 0.02). To understand how CXCR1 affects the dynamics of niche development, we globally overexpressed CXCR1 beginning at 36 hpf using a heat shock induction system and performed time lapse confocal microscopy from 52 to 72 hpf. This revealed that overexpression of CXCR1 consistently increased the CHT volume from 53 to 72 hpf compared to control (21% increase at 72 hpf, p = 0.004). These studies did not show whether CXCR1 acted directly on the vascular niche or whether CXCR1 expression in endothelial cells might induce expression of soluble factors or activate circulating cells that then cause expansion of the niche through indirect mechanisms. To address this, we created parabiotic zebrafish by fusing kdrl:mCherry embryos at 4 hpf. One half of each parabiotic animal was modified by DNA microinjection to globally overexpress CXCR1 or GFP as a control via heat shock induction at 36 and 48 hpf. The volume of the CHT was measured in each half of each parabiotic animal at 72 hpf. In control parabiotics overexpressing GFP, there was no difference in CHT volume between modified and unmodified sides of the organism. However, in parabiotics overexpressing CXCR1, the CHT of the modified side was 27% larger compared with the unmodified side (p = 0.012), consistent with our previous results and suggesting that CXCR1 acts directly on the niche in this system. We then asked whether this volume change could affect HSPC engraftment. Parabiotic animals were created using Runx1:mCherry embryos that carry an HSPC-specific reporter transgene as “donors” and WT embryos as “recipients”. The recipient niche was modified as before to overexpress CXCR1 or GFP as a control. At 72 hpf there was no difference in HSPC colonization of donor and recipient niches when the recipient niche expressed GFP. However, when the recipient niche expressed CXCR1, there was a significant increase in HSPC colonization of the recipient niche compared to the donor niche (11.4+/-2.4 vs 19.8+/-3.5 HSPCs per CHT, p = 0.02). Taken together, these results identify a novel role for CXCL8/CXCR1 signaling in angiogenesis and HSPC biology and they provide a new example of how innate immune signaling pathways are important for stem cell function. Administration of CXCL8 to hematopoietic stem cell transplant recipients may therefore improve HSPC engraftment and clinical outcomes in patients who are being treated for hematologic malignancies. Citation Format: Bradley W. Blaser, Jessica L. Moore, Elliott Hagedorn, Brian Li, Vera Binder, Owen Tamplin, Leonard I. Zon. CXCL8/CXCR1 signaling promotes angiogenesis and hematopoietic stem and progenitor cell function [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B035.

Characterization of Drug Effect on Leukemia Cells Through Single Cell Assay With Optical Tweezers and Dielectrophoresis.

One of the greatest challenges in acute myeloid leukemia (AML) treatment is preventing relapse. Leukemia cells can hide in bone marrow niche or vascular niche. Hence, many chemical drugs cannot kill these cells. To characterize migration and adhesion properties of leukemia cells in specific niches, CXCR4/SDF- 1α signal pathway has been widely used for investigation. AMD3100 is treated as one of the most common chemical drugs that can inhibit this signal. In the current study, we particularly investigate the effect of AMD3100 on the adhesion property of leukemia cells on stromal cells by using engineering tools, namely, optical tweezers (OT) and dielectrophoresis (DEP), to probe single cell property. AMD3100 not only inhibits the CXCR4/SDF- 1α signal pathway but also reduces gene expression of CXCR4 and VLA-4 on leukemia cells. The drug also softens leukemia cells. This work provides a new way to investigate cell behavior under drug treatment. The use of combined engineering tools will benefit drug discovery and assessment for leukemia treatment.

Endothelial Cells Promote Expansion of Long-Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates.

Successful expansion of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) would benefit many HSPC transplantation and gene therapy/editing applications. However, current expansion technologies have been limited by a loss of multipotency and self‐renewal properties ex vivo. We hypothesized that an ex vivo vascular niche would provide prohematopoietic signals to expand HSPCs while maintaining multipotency and self‐renewal. To test this hypothesis, BM autologous CD34+ cells were expanded in endothelial cell (EC) coculture and transplanted in nonhuman primates. CD34+C38− HSPCs cocultured with ECs expanded up to 17‐fold, with a significant increase in hematopoietic colony‐forming activity compared with cells cultured with cytokines alone (colony‐forming unit‐granulocyte‐erythroid‐macrophage‐monocyte; p < .005). BM CD34+ cells that were transduced with green fluorescent protein lentivirus vector and expanded on ECs engrafted long term with multilineage polyclonal reconstitution. Gene marking was observed in granulocytes, lymphocytes, platelets, and erythrocytes. Whole transcriptome analysis indicated that EC coculture altered the expression profile of 75 genes in the BM CD34+ cells without impeding the long‐term engraftment potential. These findings show that an ex vivo vascular niche is an effective platform for expansion of adult BM HSPCs. Stem Cells Translational Medicine 2017;6:864–876

IFNα-mediated remodeling of endothelial cells in the bone marrow niche.

In the bone marrow, endothelial cells are a major component of the hematopoietic stem cell vascular niche and are a first line of defense against inflammatory stress and infection. The primary response of an organism to infection involves the synthesis of immune-modulatory cytokines, including interferon alpha. In the bone marrow, interferon alpha induces rapid cell cycle entry of hematopoietic stem cells in vivo. However, the effect of interferon alpha on bone marrow endothelial cells has not been described. Here, we demonstrate that acute interferon alpha treatment leads to rapid stimulation of bone marrow endothelial cells in vivo, resulting in increased bone marrow vascularity and vascular leakage. We find that activation of bone marrow endothelial cells involves the expression of key inflammatory and endothelial cell-stimulatory markers. This interferon alpha-mediated activation of bone marrow endothelial cells is dependent in part on vascular endothelial growth factor signaling in bone marrow hematopoietic cell types, including hematopoietic stem cells. Thus, this implies a role for hematopoietic stem cells in remodeling of the bone marrow niche in vivo following inflammatory stress. These data increase our current understanding of the relationship between hematopoietic stem cells and the bone marrow niche under inflammatory stress and also clarify the response of bone marrow niche endothelial cells to acute interferon alpha treatment in vivo.

Distinct vascular niches determine hematopoietic stem cell fate

Identification of cellular constituents of the hematopoietic stem cell (HSC) niche has recently been the subject of intensive investigation. To investigate the spatial localization of the HSC niches in bone marrow, we have established a whole-mount immunofluorescence imaging technique in which the 3D spatial relationships between stromal structures and HSCs in the BM can be precisely determined. The imaging assessment combined with computational simulations has uncovered a significant association between HSCs and arterioles, ensheathed exclusively by rare Nestin(bright) NG2⁺ pericytes (Nes(peri) cells), distinct from sinusoid-associated Nestin(dim) Leptin receptor (LepR)⁺ (Nes(retic)) cells which reportedly represent peri-vascular niche cells. Depletion of NG2⁺ cells using NG2-creERTM / inducible diphtheria toxin receptor (iDTR) mice changed HSC localization away from arterioles, induced HSC cycling and reduced long-term repopulation of HSCs in BM, suggesting that periarteriolar NG2⁺ cells form quiescent niches for HSCs. These results form the basis of studies that will allow us to genetically dissect the functions of distinct vascular niches. This vascular niche model, in which arterioles and sinusoids differentially regulate HSC quiescence and proliferation, respectively, have implications for the behavior of healthy HSC and may be useful in the future for evaluating the niches for cancer (leukemic) stem cells.