Splenic Hematopoietic Stem Cells Research Articles

Histopathological changes in the spleen of rats exposed to N,N-dimethylhydrazine with the following protective input of Au/Ag/Fe

Colon cancer is one of the leading causes of cancer-related deaths worldwide. Despite the growing number of studies on nanoparticles of different metals, there is still a lack of reliable information about their ability to work together as a composition and their antitumor effects. It has been established that the regulation of genes encoding proteins of the Bcl-2 family changes in cancer. Bcl-2 family proteins can be a potential target in cancer diagnosis and have a prognostic value in treating cancer with chemotherapeutic drugs. The tumor microenvironment includes myeloid suppressor cells, tumor-associated neutrophils, and tumor-associated macrophages that promote cancer progression and are derived from splenic hematopoietic stem cells and progenitor cells. The work aimed to ascertain histological changes and expression of Bcl-2 family proteins in the spleen of rats with N,N-dimethylhydrazine-induced carcinogenesis followed by the protective administration of Au/Ag/Fe nanometal composition. The study was conducted on 72 outbred white male rats. Colon adenocarcinoma was modelled by N,N-dimethylhydrazine hydrochloride administration for 30 weeks. Animals received an aqueous dispersion of Au/Ag/Fe nanoparticles intragastrically once a day for 21 days at a dose of 0.842 mg Ag/0.0526 mg Fe/1.625 μg Au per 1 kg of rat body weight. Paraffin sections of the spleen were stained with hematoxylin and eosin. For immunohistochemical analysis, sections were stained with rabbit monoclonal antibodies to Bcl-2. Under conditions of 30-week exposure to N,N-dimethylhydrazine hydrochloride, blood circulation disorders, stasis, thrombosis, reduction in the size of white pulp lymphatic nodules and loss of their zonation were histologically proved in the rat's spleen. The development of sclerotic processes in the organ was noted. It was indicated that using metal nanoparticles under N,N-dimethylhydrazine hydrochloride-induced colon carcinogenesis leads to less evidenced morphological manifestations of structural changes in the spleen. Immunohistochemically, the expression of Bcl-2 protein in the white pulp of the spleen of animals subjected to correction was lower than in animals without the corrective effect of the composition of nanoparticle metals. Administration of the Au/Ag/Fe metal nanoparticle composition to intact white rats does not cause pathological changes in the spleen morphology. The structural components of the red and white pulp match the typical histological structure of the organ.

Increased iron uptake by splenic hematopoietic stem cells promotes TET2-dependent erythroid regeneration

Hematopoietic stem cells (HSCs) are capable of regenerating the blood system, but the instructive cues that direct HSCs to regenerate particular lineages lost to the injury remain elusive. Here, we show that iron is increasingly taken up by HSCs during anemia and induces erythroid gene expression and regeneration in a Tet2-dependent manner. Lineage tracing of HSCs reveals that HSCs respond to hemolytic anemia by increasing erythroid output. The number of HSCs in the spleen, but not bone marrow, increases upon anemia and these HSCs exhibit enhanced proliferation, erythroid differentiation, iron uptake, and TET2 protein expression. Increased iron in HSCs promotes DNA demethylation and expression of erythroid genes. Suppressing iron uptake or TET2 expression impairs erythroid genes expression and erythroid differentiation of HSCs; iron supplementation, however, augments these processes. These results establish that the physiological level of iron taken up by HSCs has an instructive role in promoting erythroid-biased differentiation of HSCs.

Deciphering the differential impact of thrombopoietin/MPL signaling on hematopoietic stem/progenitor cell function in bone marrow and spleen

Thrombopoietin (TPO) and its receptor MPL play crucial roles in hematopoietic stem cell (HSC) function and platelet production. However, the precise effects of TPO/MPL signaling on HSC regulation in different hematopoietic niches remain unclear. Here, we investigated the effects of TPO/MPL ablation on marrow and splenic hematopoiesis in TPO-/- and MPL-/- mice during aging. Despite severe thrombocytopenia, TPO-/- and MPL-/- mice did not develop marrow failure during a 2-year follow-up. Marrow and splenic HSCs exhibited different responses to TPO/MPL ablation and exogenous TPO treatment. Splenic niche cells compensated for marrow HSC loss in TPO-/- and MPL-/- mice by upregulating CXCL12 levels. These findings provide new insights into the complex regulation of HSCs by TPO/MPL and reveal a previously unknown link between TPO and CXCL12, two key growth factors for HSC maintenance. Understanding the distinct regulatory mechanisms between marrow and spleen hematopoiesis will help to develop novel therapeutic approaches for hematopoietic disorders.

Deciphering the Differential Impact of Thrombopoietin/MPL Signaling on Hematopoietic Stem Cell Function in Bone Marrow and Spleen

Introduction Thrombopoietin (TPO) and its receptor MPL are key regulators of hematopoietic stem cell (HSC) function. In humans, ablation of either TPO or MPL leads to bone marrow failure in the early years of life. In contrast, mice lacking TPO or MPL display marked thrombocytopenia and severely reduced HSC numbers but manage to maintain normal survival up to one year of age. In this study, we investigated whether these mice eventually develop marrow failure with longer follow up, with a specific focus on understanding the effects of TPO/MPL signaling on HSC regulation in different hematopoietic niches. Methods Marrow and spleen hematopoiesis in the TPO -/- mice and MPL -/- mice were examined during a 2-yr follow-up. Results At 2 yrs of age, TPO -/- and MPL -/- mice exhibited not only a severely decreased platelet count (TPO -/- vs. MPL -/- vs. control: 38 vs. 107 vs. 1255 x 10 9/L) but also a moderate reduction in neutrophil count compared to age-matched wild-type controls (TPO -/- vs. MPL -/- vs. control: 1.1 vs. 1.7vs.3.3 x 10 3/ul). Femur marrow cell count or spleen cell count was not significantly different between TPO -/- or MPL -/- mice and control mice. There was no significant difference in overall survival between the TPO -/- or MPL -/- mice and wild-type controls. Marrow Lin -cKit +Sca1 +CD150 +CD48 - HSC cell numbers were significantly decreased in young (4-6mo) TPO -/- and MPL -/- mice (~4-fold) compared to age-matched control mice. This HSC loss was further worsened in old (~24mo) TPO -/- (25-fold) and MPL -/- (12-fold) mice compared to control mice. In contrast, there was no significant difference in splenic HSC cell numbers between young TPO -/- or MPL -/- mice and control mice, with only a moderate decrease of HSCs in old TPO -/- and MPL -/- (~4-fold) mice compared to control mice. I n vivo using BrdU labeling revealed an increased proliferation of marrow HSCs in TPO -/- and MPL -/- mice compared to age-matched control mice. In contrast, there was no significant increase in splenic HSC proliferation in TPO -/- or MPL -/- mice. These findings suggest that TPO/MPL signaling has distinct effects on both the number and the function of HSCs in the marrow and spleen. Competitive repopulation assays revealed that HSCs from 2yr old TPO -/- or MPL -/- marrow failed to engraft the recipient mice at 12 weeks post-transplantation. We also conducted competitive repopulation assays in which spleen cells from old TPO -/- mice (CD45.2) were injected together with spleen cells from 2yr old wild-type competitor mice (CD45.1) into lethally irradiated wild-type recipients (CD45.1). At 12 days after transplantation, the TPO -/- spleen donor showed comparable engraftment in the Lin -cKit +Sca1 + stem/progenitor cell compartment, similar to the wild-type competitor spleen donor, suggesting that at least the short-term engraftment capacity of the TPO -/- spleen donor was not impaired. We are currently conducting further investigation to assess the long-term repopulation capacity of TPO -/- spleen HSCs. To investigate how TPO/MPL ablation impacts the hematopoietic niche in the marrow and spleen, we used quantitative flow cytometry and/or fluorescence imaging to examine these niche cells and their expression of niche factors. First, while there was no significant difference in the marrow vascular area (measured by in vivo VE-cadherin labeling) between TPO -/- or MPL -/- mice and control mice, we observed a significant increase in the splenic vascular area in TPO -/- and MPL -/- mice. Second, CXCL12 levels were significantly decreased in marrow endothelial cells (ECs; CD45 -CD31 +) from TPO -/- and MPL -/- mice compared to control mice; in contrast, CXCL12 levels were significantly increased in splenic ECs from TPO -/- and MPL -/- mice. Similar results were also observed with marrow and splenic perivascular stromal cells (CD45 -CD31 -Ter119 -Sca1 -). Therefore, TPO/MPL ablation enhanced both the quantity and quality of the splenic hematopoietic niche, which could contribute to the maintenance of splenic hematopoiesis in TPO -/- and MPL -/- mice. Last, consistent with findings from the TPO/MPL ablated mice, exogenous TPO treatment increased marrow HSC proliferation but did not alter splenic HSC proliferation. In addition, TPO treatment increased CXCL12 expression in marrow ECs, but decreased CXCL12 levels in splenic ECs. Taken together, our study revealed the complexity of TPO/MPL signaling in HSC regulation which differs between the marrow and spleen

Conditionally Pathogenic Genetic Variants of a Hematopoietic Disease-Suppressing Enhancer

Human genetic variants are classified based on potential pathogenicity to guide clinical decisions. However, mechanistic uncertainties often preclude definitive categorization. Germline coding and enhancer variants within the hematopoietic regulator GATA2 create a bone marrow failure and leukemia predisposition (Soukup and Bresnick, 2020). The conserved murine enhancer (+9.5) promotes hematopoietic stem cell (HSC) genesis, and a single-nucleotide human variant in an Ets motif attenuates chemotherapy- and transplantation-induced hematopoietic regeneration in a cell-autonomous manner (Soukup et al., 2019). Since an epigenetically-silenced normal allele can exacerbate phenotypes of a pathogenic heterozygous variant, we engineered a bone marrow failure model harboring the Ets motif variant and a severe E-box:GATA enhancer mutation on the second allele (CH) that renders the enhancer chromatin inaccessible, to determine how perturbations of the hematopoietic system trigger pathogenesis.Given the propensity of GATA2 deficiency syndrome patients to infection, we asked if CH mice could mount an appropriate response to the bacterial cell wall component LPS. In WT, LPS acts on HSCs to induce inflammatory signaling and proliferation (Caiado et al., 2021). Twenty-four hours after LPS (0.5 mg/kg) administration, CH LSK levels were 3.1-fold lower than WT (p = 0.009). Immunophenotypic LT-HSCs (LSKCD150 +CD34 -) were 1.9-fold lower in treated CH vs. WT mice (p = 0.019). LPS reduced all LSK subpopulations: CD34 -CD150 -, CD34 +CD150 -, and CD34 +CD150 + were 3.4-, 5.7-, and 5.3-fold lower, respectively (p = 0.007, 0.029, and 0.006). Thus, the CH mutation attenuated acute inflammation-induced HSPC expansion.G-CSF is among the cytokines/chemokines induced by infection (Quinton et al., 2002). As G-CSF is used to mobilize bone marrow HSCs for collection prior to transplantation, we tested the following models: 1) +9.5 mutation abrogates HSPC expansion and/or mobilization in response to diverse stimuli; 2) +9.5 mutation permits expansion/mobilization, yet compromises function; 3) HSPC expansion, mobilization and function are +9.5 mutation-insensitive. After 8 doses of G-CSF, circulating neutrophil numbers increased 2.6-fold in WT (p = 0.003), while neutrophils did not increase in the CH mice. G-CSF treatment increased bone marrow HSCs 2.6-fold in WT mice, whereas the CH mutation abrogated this response (p < 0.0001). As a metric of mobilization, splenic HSCs were quantified. After G-CSF treatment, WT splenic HSCs expanded 11-fold (p < 0.0001), while CH HSCs were unchanged. To functionally analyze mobilized HSPCs, colony forming unit (CFU) assays were conducted with peripheral blood. CFU increased 11-fold in WT blood post-treatment (p < 0.0001), and CH abrogated the increase.To test whether G-CSF signaling is impacted by the CH mutation, we asked whether G-CSF treatment of bone marrow ex vivo induced STAT3 phosphorylation using a phospho-flow cytometry assay. While G-CSF induced pSTAT3 8.5-fold in WT Lin -Kit + cells (p < 0.0001), the induction was attenuated to 4.9-fold in CH (p = 0.003). Signaling in Gr1 + granulocytes was indistinguishable between genotypes (p = 0.999). Thus, the CH mutation attenuated G-CSF-induced signaling and HSPC mobilization, abrogated HSPC expansion, and compromised HSPC function.The Gata2 CH bone marrow failure model is characterized by relatively normal developmental hematopoiesis and adult steady-state hematopoiesis, yet the mice are vulnerable to stressors. As the CH mice were hypersensitive to myeloablation and acute inflammation and exhibited an attenuated response to a clinical HSC-mobilizing agent, GATA2-driven regenerative hematopoiesis mitigates damage resulting from chemotherapy and inflammation and promotes G-CSF-induced HSC mobilization. As the enhancer variant controlled HSC responsiveness to a therapeutically important HSC-mobilizing drug, these results link non-coding genome variation with mobilization efficacy. Based on the prominent GATA2 activities described herein, we are investigating how the full ensemble of macromolecular components constituting the GATA2 regulatory network generate a vital protective system conferring resilience to the hematopoietic system in the context of diverse stressors and whether genetic variants in genes encoding components of these pathways resemble or differ from those of GATA2 variants. DisclosuresNo relevant conflicts of interest to declare.

3092 – STABLE COLONY STIMULATING FACTOR 1 FUSION PROTEIN TREATMENT INCREASES THE HEMATOPOIETIC STEM CELL POOL AND ENHANCES THEIR MOBILIZATION IN MICE.

Prior chemotherapy and/or underlying morbidity commonly leads to poor mobilization of hematopoietic stem cells (HSC) for autologous transplantation in cancer patients. Increasing the number of available HSC prior to mobilization is a potential strategy to overcome this deficiency. Bone marrow (BM) macrophages regulate HSC niches and promote engraftment of donor HSC post-transplant. In this study, we examined if manipulating macrophages using a modified long-acting Fc fusion protein of CSF1 could influence HSC niches to expand the HSC pool. Acute CSF1-Fc treatment (4 x daily injection) in C57Bl/6 mice induced a robust systemic myeloproliferative response that peaked 1 week post the initial injection. BM and spleen homeostasis were disrupted during peak myelopoiesis, including supressed lymphopoiesis and temporary engraftment of BM hematopoietic stem and progenitor cell (HSPC) in liver. CSF1-Fc effects were transient as by 2 weeks post first CSF1-Fc injection, overall haematopoiesis had normalized, but the number of HSC in BM (1.6 fold), and particularly in spleen (5.9 fold), were significantly elevated compared to controls. This was not due to direct CSF1-Fc actions on HSC as multiple experimental approaches confirmed that HSC and multipotent progenitors do not express CSF1-receptor. The increase in splenic HSC corresponded with a sustained phenotype shift in red pulp macrophages characterized by expression of the BM HSC niche macrophage marker CD169. We observed minimal evidence of in situ macrophage proliferation, indicating specialised adaptation of monocyte-derived macrophages to the CSF1-Fc altered splenic environment. Importantly, CSF1-Fc pre-treatment enhanced HSC mobilization (2.5 fold) into blood after G-CSF treatment when compared to controls. Competitive transplantation demonstrated the CSF1-Fc + G-CSF mobilized blood graft had improved reconstitution potential with no leukocyte lineage bias. Overall, the data suggest that CSF1-Fc treatment resulted in sustained adaptation of resident macrophages with improved capability to support extramedullary HSC niches resulting in a systemic increase in the absolute number of HSC. Therefore, conditioning patients with CSF1 could be a potential therapeutic strategy to overcome poor mobilization and subsequently improve autologous transplantation outcomes.

Functional Interaction of BRCA1 and CREBBP in Murine Hematopoiesis.

SummaryBoth BRCA1 and CREBBP are tumor suppressor genes that are important for hematopoiesis. We have previously shown that mouse Brca1 is essential for hematopoietic stem cell (HSC) viability. In contrast to Brca1 deficiency, which results in pancytopenia, we report here that Crebbp deficiency results in myeloproliferation associated with an increase of splenic HSCs as well as a lethal systemic inflammatory disorder (LD50 = 86 days). To investigate the interaction of these two proteins in hematopoiesis, we generated double Crebbp/Brca1 knockout mice (DKOs). To our surprise, DKOs had accelerated bone marrow failure compared with Brca1-deficient mice and this was associated with an even shorter lifespan (LD50 = 88.5 versus 33 days). Furthermore, Crebbp or Brca1 heterozygosity influenced the hematopoietic phenotype associated with complete deficiency of Brca1 or Crebbp, respectively. We also observed lower BRCA1 protein levels in hematopoietic tissues when CREBBP is absent. Collectively, these data suggest Crebbp and Brca1 functionally interact to maintain normal hematopoiesis.

The Regulation of Hematopoietic Stem Cells and Erythropoiesis

Hematopoietic stresses mobilize hematopoietic stem cells (HSCs) from the bone marrow to the spleen and induce extramedullary hematopoiesis (EMH), including erythropoiesis. We assessed the sources of the key niche factors, SCF and CXCL12, in the mouse spleen after EMH induction by cyclophosphamide plus granulocyte colony-stimulating factor, blood loss, or pregnancy. In each case, Scf was expressed by endothelial cells and Tcf21+ stromal cells, primarily around sinusoids in the red pulp, while Cxcl12 was expressed by a subset of Tcf21+ stromal cells (Nature 527:466, 2015). EMH induction markedly expanded the Scf-expressing endothelial cells and stromal cells by inducing their proliferation. Most splenic HSCs were adjacent to Tcf21+ stromal cells in red pulp. Conditional deletion of Scf from spleen endothelial cells or Scf or Cxcl12 from Tcf21+ stromal cells severely reduced spleen EMH and reduced blood cell counts without affecting bone marrow hematopoiesis. Endothelial cells and Tcf21+ stromal cells thus create a perisinusoidal EMH niche in the spleen, which is necessary for the physiological response to diverse hematopoietic stresses.During pregnancy, the blood volume increases relatively quickly, requiring increased erythropoiesis to maintain red blood cell counts. We discovered that HSCs in female mice divide significantly more frequently than in males as a result of estrogen/estrogen receptor signaling in HSCs (Nature 505:555). Estrogen levels increase during pregnancy, increasing HSC division, HSC frequency, cellularity, and erythropoiesis in the spleen. Conditional deletion of Estrogen Receptor α from HSCs reduced HSC division in female, but not male, mice and attenuated the increases in HSC division, HSC frequency, and erythropoiesis during pregnancy. Treatment of mice with estradiol induces HSC proliferation in the bone marrow but not mobilization. Treatment with the alternative ERα ligand, 27-hydroxycholesterol (27HC), induces ERα-dependent HSC mobilization and EMH but not HSC division in the bone marrow (JCI 127:3392). During pregnancy, 27HC levels increased in hematopoietic stem/progenitor cells as a result of Cyp27a1, a cholesterol hydroxylase. Cyp27a1-deficient mice have significantly reduced 27HC levels, HSC mobilization, and EMH during pregnancy. Two different ERα ligands, estradiol and 27HC, thus work together to promote EMH during pregnancy.We have also examined the bone marrow to better understand the niche for erythropoiesis. HSCs in the bone marrow are maintained in a perivascular niche created by Leptin Receptor+ (LepR) stromal cells and endothelial cells. An important question is why LepR+ cells are one hundred times more frequent than HSCs. We discovered that SCF from LepR+ cells is also necessary for the maintenance of many c-kit+ restricted hematopoietic progenitors. Conditional deletion of Scf from LepR+ cells depleted CMPs, CLPs, GMPs, MEPs, PreMegEs, and CFU-Es, depleting myeloid and erythroid cells from the blood. This was not a consequence of HSC depletion as many other restricted progenitors were unaffected. Moreover, conditional deletion of Scf from endothelial cells depleted HSCs, but not restricted progenitors. Imaging showed that early erythroid progenitors were closely associated with perisinusoidal LepR+ cells. Our data reveal cellular specialization within the niche: SCF from LepR+ cells is broadly required by HSCs and c-kit+ restricted progenitors while SCF from endothelial cells is required only by HSCs. Erythroid progenitors depend upon SCF synthesized by LepR+ cells in perisinusoidal niches. DisclosuresMorrison:OncoMed Pharmaceuticals: Equity Ownership; GI Therapeutics: Consultancy, Equity Ownership; Frequency Therapeutics: Consultancy.

Splenic hematopoietic stem cells display a pre-activated phenotype.

Splenic hematopoiesis is crucial to the pathogenesis of diseases including myocardial infarction and atherosclerosis. The spleen acts as a reservoir of myeloid cells, which are quickly expelled out in response to acute inflammation. In contrast to the well-defined bone marrow hematopoiesis, the cellular and molecular components sustaining splenic hematopoiesis are poorly understood. Surprisingly, we found that, unlike quiescent bone marrow hematopoietic stem cells (HSC), most of splenic HSC are in the G1 phase in C57BL/6 mice. Moreover, splenic HSC were enriched for genes involved in G0-G1 transition and expressed lower levels of genes responsible for G1-S transition. These data indicate that, at steady state, splenic HSC are pre-activated, which may expedite their cell cycle entry in emergency conditions. Consistently, in the acute phase of septic shock induced by LPS injection, splenic HSC entered the S-G2-M phase, whereas bone marrow HSC did not. Mobilization and transplantation experiments displayed that bone marrow HSC, once in the spleen, acquired cell cycle status similar to splenic HSC, strongly suggesting that the splenic microenvironment plays an important role in HSC pre-activation. In addition, we found that myeloid translocation gene 16 (Mtg16) deficiency in C57BL/6 mice resulted in significantly increased S-G2-M entry of splenic but not bone marrow HSC, suggesting that Mtg16 is an intrinsic negative regulator of G1-S transition in splenic HSC. Altogether, this study demonstrates that compared to bone marrow, splenic HSC are in a pre-activated state, which is driven by extracellular signals provided by splenic microenvironment and HSC intrinsic factor Mtg16.

Notch Downregulation and Extramedullary Erythrocytosis in Hypoxia-Inducible Factor Prolyl 4-Hydroxylase 2-Deficient Mice.

Erythrocytosis is driven mainly by erythropoietin, which is regulated by hypoxia-inducible factor (HIF). Mutations in HIF prolyl 4-hydroxylase 2 (HIF-P4H-2) (PHD2/EGLN1), the major downregulator of HIFα subunits, are found in familiar erythrocytosis, and large-spectrum conditional inactivation of HIF-P4H-2 in mice leads to severe erythrocytosis. Although bone marrow is the primary site for erythropoiesis, spleen remains capable of extramedullary erythropoiesis. We studied HIF-P4H-2-deficient (Hif-p4h-2gt/gt) mice, which show slightly induced erythropoiesis upon aging despite nonincreased erythropoietin levels, and identified spleen as the site of extramedullary erythropoiesis. Splenic hematopoietic stem cells (HSCs) of these mice exhibited increased erythroid burst-forming unit (BFU-E) growth, and the mice were protected against anemia. HIF-1α and HIF-2α were stabilized in the spleens, while the Notch ligand genes Jag1, Jag2, and Dll1 and target Hes1 became downregulated upon aging HIF-2α dependently. Inhibition of Notch signaling in wild-type spleen HSCs phenocopied the increased BFU-E growth. HIFα stabilization can thus mediate non-erythropoietin-driven splenic erythropoiesis via altered Notch signaling.

F4/80 Identifies a Subset of Non-Mobilizable Bone Marrow HSCs Involved in Stress-Induced Hematopoiesis

Hematopoietic stem cell (HSCs) are commonly isolated by using cell surface markers in order to study their hierarchy and functional properties. However, even with the most rigorous methods of isolation, subsets of HSCs likely exhibit functional heterogeneity. We have found that F4/80, an adhesion G-protein coupled receptor well known as a macrophage marker, was expressed on an HSC subpopulation (50-60% of Lin− Sca1− cKit− CD150+ CD34− CD135− cells) in the bone marrow. Interestingly, F4/80 was not expressed on HSCs that have egressed in the blood and on only a small fraction (3%) of splenic HSCs. To evaluate the function of the HSC subset expressing F4/80, we transplanted competitively 200 F4/80+ or F4/80− HSCs using CD45.1/2 congenic system. We found that F4/80+ HSCs exhibited a lower engraftment potential compared to F4/80− HSCs at 16 weeks after transplantation (19.7±4.8% and 37.1±4.5% donor contribution, respectively, P=0.025), although both HSC subsets were able to sustain mixed chimerism for myeloid, B and T cells without significant alteration in lineage bias. Since F4/80+ HSCs were not found in extramedullary tissues (blood or spleen), we tested whether they could be mobilized following G-CSF or CXCR4 antagonist (AMD3100) treatment. Interestingly, F4/80+ HSCs were still retained in the bone marrow after either G-CSF- or AMD3100-induced mobilization whereas F4/80− HSCs were mobilized efficiently. However, the frequency of bone marrow F4/80+ HSCs was reduced in G-CSF-treated animals (3.3-fold, p<0.0001) while the F4/80− HSC frequency was increased (2.2-fold, p<0.0001). F4/80 could act directly to retain HSCs in the bone marrow microenvironment or it could mark a non-mobilizable pool of stem cells. To test the later possibility, we crossed transgenic mice expressing Cre recombinase knocked in the CD169 locus, a marker of bone marrow macrophage, with ROSA26-loxP-stop-loxP-tdTomato (CD169/tomato). We found that CD169/tomato selectively labelled a large fraction of F4/80+ HSCs (31.7±8.4%) by contrast to F4/80− HSCs which were, by and large, not labelled (2.1±1.2%). We next induced HSC mobilization with G-CSF. Strikingly, we found that in a manner similar to F4/80+ HSCs, CD169/tomato+ HSCs were not mobilized in the blood while their numbers were reduced in the bone marrow after G-CSF (5-fold, p=0.014). Since macrophage depletion can induce HSC mobilization (Chow et al., JEM 2011), we tested the effect of macrophage depletion using clodronate liposomes. Interestingly, F4/80+ HSCs in wild-type mice or CD169/tomato+ HSCs were not mobilized following macrophage depletion but were depleted from the bone marrow while F4/80− HSCs underwent a 3-fold expansion. The reduced HSC numbers in marrow upon mobilization by G-CSF suggested a role in survival or proliferation. To evaluate proliferation status at steady state, we performed cell cycle analyses and BrdU incorporation assay which revealed that F4/80+ HSCs significantly more proliferative than F4/80− HSCs either by cell cycle analyses (3.7-fold increase in non-G0 phase, p=0.024) or by BrdU incorporation (2-fold increase, p=0.026). We next assessed the impact of 5-flurouracil (5FU) administration a chemotherapeutic agent that kills cycling cells and induce stress hematopoietic recovery. While both F4/80+ and F4/80− HSCs were depleted 4 days after 5FU treatment, we observed that F4/80+ HSCs expanded dramatically compared to F4/80− HSCs (10-fold and 4.5-fold over F4/80− HSCs at 8 days and 12 days post-5FU, respectively) during recovery phase. Taken together, these results identify F4/80+ HSCs as a strictly resident subset of bone marrow HSCs involved in rapid recovery after hematopoietic stress. DisclosuresFrenette:PHD Biosciences: Research Funding; GSK: Research Funding; Pfizer: Consultancy.

Compromised emergency hematopoiesis in obesity requires TLR4

Abstract Following acute injury/infection, bone marrow (BM) precursors must responsively redirect from steady-state to emergency hematopoiesis. Prospective and retrospective studies establish obesity as risk factor for infection but the mechanisms are poorly understood. Here, we show in a murine model of obesity that moderate changes in steady-state BM hematopoiesis become exaggerated during hematopoietic duress. At steady-state, diet-induced obese mice have a 30% decrease in BM hematopoietic stem cells (HSCs) with a parallel increase in splenic HSCs. HSCs are functionally competent in vitro and following competitive transfer in vivo. BM lymphoid and myeloid progenitors in obese mice are numerically intact but the transcriptional profiles and differentiation potential are strikingly myeloid-biased, accompanied by myeloid skewing of peripheral blood. Responses to acute hematopoietic challenge are deficient. Emergency hematopoiesis following chemotherapeutic ablation is reduced 60% in obesity. Likewise, emergency myelopoiesis in response to endotoxin is characterized by accumulation of immature rather than mature myeloid cells in obese mice. The obesity-associated inflammatory environment plays a key role as transfer of obese BM to lean recipients restores emergency immune response to chemotherapeutic challenge. Moreover, mice deficient in toll-like receptor 4 (TLR4), a central regulator of inflammation, retain robust hematopoietic rebound responses despite diet-associated weight gain. Thus, in obesity, moderate changes in steady-state blood cell production mask major defects in hematopoietic stress responses, via a TLR4-dependent mechanism.

Notch signaling directs splenic resident hematopoietic stem cell fate decisions

Abstract Adult hematopoiesis occurs primarily in the bone marrow (BM), generating mature blood cells as well as maintaining a heterogeneous pool of self-renewing hematopoietic stem cells (HSCs). However, studies suggest that the BM may not be the only site of adult hematopoiesis: during times of BM hematopoietic distress stemming from radiation or disease, the spleen can independently reconstitute the blood. Given that the spleen does indeed contain a small, resident population of lineage-sca1+ckit+CD48-CD150+ long-term (LT)-HSCs with potent reconstitution ability, we investigated the peripheral blood cell contribution of these extra-medullary HSCs by transplanting UBC-GFP Tg reporter splenic fragments into a splenectomized syngenic wildtype mouse and tracking the blood cell production over time. While the spleen gives rise to both mature myeloid and lymphoid cells, we find that the proportion is skewed in favor of T cells. We see spleen-derived donor HSCs generating CD4+CD8+ double positive thymocytes for over five months post-transplant, suggesting that this peripheral T cell population derives from a long-lived stem or progenitor cell resident in the spleen. RNA sequencing demonstrates that splenic LT-HSCs are more responsive to Notch signaling than BM LT-HSCS, as indicated by increased levels of Hes1 transcription and decreased levels of the granulocyte/monocyte (GM) lineage priming genes Mpo, Gfi1, and Fcgr2b. Taken together with our finding that splenic HSCs have higher levels of cytoplasmic Notch2, we suggest that the Notch2-Hes1 axis is simultaneously repressing GM priming and enforcing T cell programming even among the most immature HSCs resident in the spleen.

Targeting splenic hematopoietic stem cells in cardiovascular disease

Inflammation plays a crucial role in cardiovascular disease. For example, inflammatory monocytes, after being recruited to atherosclerotic plaques, secrete proteinases that erode the fibrous cap. This may lead to plaque rupture and subsequent myocardial infarction (MI). Patients with acute myocardial infarction have elevated levels of blood leukocytes [1]. Peripheral blood leukocyte count after MI correlates with in-hospital mortality, underscoring role of inflammatory cells in post-MI myocardial remodeling. The spleen provides steady flow of leukocytes to the infarct and atherosclerotic plaques. Since monocyte turnover at the sites of inflammation is very high [2], hematopoietic stem and progenitor cells (HSPC) differentiate into myeloid cells to keep up with the increased demand. MI drives splenic HSPC into the cell cycle [3] and induces their lineage differentiation. Subsequent imaging studies [4, 5] suggested splenocyte proliferation after acute myocardial infarction in patients. Although splenic myelopoiesis exaggerates inflammation in cardiovascular disease, the components of splenic hematopoietic niche are not well understood. Since macrophages play a major role in bone marrow HSC maintenance, we investigated if macrophages regulate hematopoiesis in the spleen [6]. Toward this end, we developed lipidoid nanoparticles containing siRNA against CD115, the receptor for MCS-F, which is involved in macrophage differentiation, proliferation and survival. CD115 knockdown significantly reduced splenic macrophage numbers in mice injected with the Toll like receptor ligand lipopolysaccharide. Concomitantly, splenic HSC, HSPC and granulocyte macrophage progenitor numbers were diminished after knocking down CD115. Levels of VCAM-1, an HSC retention factor, were reduced in the spleen; however, HSC proliferation and apoptosis were unaltered, indicating impaired retention of splenic HSC after CD115 knockdown. Reduced splenic retention was in line with elevated HSPC levels in the blood after siCD115 treatment. Additionally, we found similar diminished splenic HSC numbers after depletion of splenic macrophages in CD169-iDTR mice with diphtheria toxin, bolstering the hypothesis that macrophages maintain splenic HSC. CD115 knockdown curtailed myeloid cell supply to the infarct in mice on day 4 after coronary ligation. Furthermore, the treatment also abated inflammation in atherosclerotic lesions in ApoE−/− mice fed with high fat diet. This reduction of inflammation was likely a consequence of mitigated extramedullary myelopoiesis due to impaired HSC maintenance after CD115 knockdown. In agreement with reduced inflammation, the treatment reduced plaque size and necrotic core area, and thickened fibrous cap. These are features of stable atherosclerotic plaques. Since VCAM-1 was the only HSC retention factor that was downregulated in splenocytes after CD115 knockdown, we investigated which hematopoietic cells express VCAM-1. Out of all hematopoietic cells tested, only macrophages expressed VCMA-1 at high levels. An adoptive transfer experiment revealed that splenic HSPC reside in close proximity with VCAM-1+ macrophages. In fact, more than 90% of HSPC reside within 10 μm distance from this macrophage subset. These data indicate that VCAM-1+ macrophages maintain splenic HSC. To investigate this further, we formulated siRNA against VCAM-1 in lipidoid nanoparticles which enrich in macrophages but not endothelial cells. VCAM-1 knockdown in macrophages significantly reduced myelopoiesis and HSC numbers in the spleen. Of note, the treatment did not change HSC numbers in the bone marrow, indicating involvement of other cell types and redundant mechanisms of retention for bone marrow HSC maintenance. Further, macrophage-specific VCAM-1 knockdown reduced inflammation in atherosclerosis and improved characteristics of stable atherosclerotic plaques. In summary, the study suggests that VCAM-1+ macrophages are important components of splenic HSC niche. In absence of VCAM-1 or splenic macrophages, HSC depart from the spleen, resulting in decreased myelopoiesis after MI and in mice with atherosclerosis. We previously showed that MI-induced splenic HSPC proliferation is stem cell factor-dependent [3]. Endothelial cells are major source of stem cell factor, which maintain HSC in the bone marrow [7]. However, it is not known if SCF secreted by splenic endothelial cells after MI triggers myelopoiesis. The bone marrow HSC niche is a complex microenvironment and orchestrated by different cells such as nestin+ mesenchymal stem cell, endothelial cells and regulatory T cells. It remains to be investigated which splenic cells besides macrophages maintain HSC.

Macrophages retain hematopoietic stem cells in the spleen via VCAM-1

Splenic myelopoiesis provides a steady flow of leukocytes to inflamed tissues, and leukocytosis correlates with cardiovascular mortality. Yet regulation of hematopoietic stem cell (HSC) activity in the spleen is incompletely understood. Here, we show that red pulp vascular cell adhesion molecule 1 (VCAM-1)(+) macrophages are essential to extramedullary myelopoiesis because these macrophages use the adhesion molecule VCAM-1 to retain HSCs in the spleen. Nanoparticle-enabled in vivo RNAi silencing of the receptor for macrophage colony stimulation factor (M-CSFR) blocked splenic macrophage maturation, reduced splenic VCAM-1 expression and compromised splenic HSC retention. Both, depleting macrophages in CD169 iDTR mice or silencing VCAM-1 in macrophages released HSCs from the spleen. When we silenced either VCAM-1 or M-CSFR in mice with myocardial infarction or in ApoE(-/-) mice with atherosclerosis, nanoparticle-enabled in vivo RNAi mitigated blood leukocytosis, limited inflammation in the ischemic heart, and reduced myeloid cell numbers in atherosclerotic plaques.

Infection mobilizes hematopoietic stem cells through cooperative NOD-like receptor and Toll-like receptor signaling.

Adult hematopoietic stem cells (HSCs) are maintained in specialized niches within the bone marrow under steady-state conditions and mobilize for extramedullary hematopoiesis during periods of stress such as bacterial infections. However, the underlying mechanisms are unclear. We show that systemic infection of mice with Escherichia coli, commonly associated with bacteremia in humans, mobilizes functional HSCs to the spleen. Accumulation of splenic HSCs (CD150+CD48-Lin(-/low)Sca1+cKit+) was diminished in TLR4-deficient and RIPK2-deficient mice, implicating TLRs and cytosolic NOD1/NOD2 signaling in the process. Accordingly, dual stimulation of NOD1 and TLR4 in radio-resistant cells alone was sufficient to mobilize HSCs, while TLR4 expression on HSCs was dispensable. Mechanistically, TLR4 and NOD1 synergistically induced granulocyte colony-stimulating factor (G-CSF), which was required for extramedullary HSC accumulation. Mobilized HSCs and progenitor cells gave rise to neutrophils and monocytes and contributed to limiting secondary infection.

Pten loss in the bone marrow leads to G-CSF–mediated HSC mobilization

The phosphatase and tumor suppressor PTEN inhibits the phosphoinositol-3-kinase (PI3K) signaling pathway and plays a key role in cell growth, proliferation, survival, and migration. Pten conditional deletion using MxCre or Scl-CreER(T) leads to splenomegaly and leukemia formation, which occurs after the relocation of normal hematopoietic stem cells (HSCs) from the bone marrow to the spleen. Unexpectedly, dormant HSCs in the bone marrow do not enter the cell cycle upon Pten loss, they do not lose self-renewal activity, and they are not exhausted. Instead, Pten deficiency causes an up-regulation of the PI3K pathway in myeloid cells, but not in HSCs. Strikingly, myeloid cells secrete high levels of G-CSF upon Pten loss, leading to the mobilization of HSCs from the bone marrow and accumulation in the spleen. After deletion of Pten in mice lacking G-CSF, the splenomegaly, myeloproliferative disease, and splenic HSC accumulation are rescued. Our data show that although PTEN has little if any role in normal HSCs, it is essential to prevent overt G-CSF production by myeloid and stromal cells which otherwise causes HSCs to relocate to the spleen followed by lethal leukemia initiation.

This Niche Is a Maze; An Amazing Niche

The cellular identity of niche cells that regulate hematopoietic stem cell (HSC) self-renewal and differentiation has been debated for several years. Two recent studies in Nature (Ding and Morrison, 2013; Greenbaum etal., 2013) have shed light into the bone marrow stromal subsets making CXCL12, a chemokine critical for HSC maintenance.

Mobilization as a Preparative Regimen for Hematopoietic Stem Cell (HSC) Transplantation.

Cytokines release HSC from marrow and thus facilitate their collection from blood. We performed studies in parabiotic mice to determine if microenvironmental niches are vacated when HSC are mobilized. In our initial experiments, ROSA26 (CD45.2+) and Pep3b (CD45.1+) (both C57BL6) mice were joined in parabiosis for 3, 6, 8 or 12 wks. Although their circulations were shared (approximately 50% of granulocytes in the blood of each parabiont had partner phenotype in all studies), few HSC engrafted in partner marrow. Specifically, 1.0–2.5 % of marrow HSC had a partner phenotype, as determined by the transplantation of marrow cells into irradiated secondary recipients (Blood 102,1249,2003). Also, few marrow granulocytes and CFU-GM had a partner phenotype, implying that marrow functions as an intact compartment in which resident HSC give rise to progenitors then mature cells. In contrast, in the spleen, 1.5–3.6% of HSC, yet 38–55% of granulocytes and CFU-GM had a partner phenotype. We then treated each parabiont with one cycle of hG-CSF (25 ug/kg) and hSCF (200 ug/kg) sq qd x 4d on days 17–20 of parabiosis and examined the marrow at 6 weeks (day 42). 10.1±6.2(SD)% of HSC had partner phenotype (p=0.02). When 3 cycles of cytokines were administered beginning days 17, 24 and 31, 13.9±9.0% of marrow HSC had partner phenotype at 6 wks of parabiosis (p=0.01). Experiments were then repeated with AMD3100 (a SDF-1/CXCR4 axis antagonist, gift of AnorMed, Langley, BC, CA; 5mg/kg/mouse sq on day 20; n=3 pairs (6 parabionts)) to demonstrate that HSC mobilization, and not replication, was responsible for these results. At 6 wks of parabiosis, 5.9±2.9% of marrow HSC, 5.7±2.3% of marrow CFU-GM and 5.6±2.8% of marrow granulocytes had partner phenotype (p=0.02), while 7.2±2.7%, 44.0±3.2% and 40.0±6.1% of splenic HSC, CFU-GM and granulocytes had partner phenotype, respectively. These data imply that HSC exited marrow, transited blood, engrafted in open niches in partner marrow, and contributed (normally) to hematopoiesis. Similar percentages of HSC also engrafted in spleen. However, splenic hematopoiesis, as at baseline, derived from CFU-GM, not HSC, engraftment. We next tested a corollary of these findings. If niches are vacated after AMD3100 administration, transplanted HSC might preferentially engraft. Pep3b mice were treated with AMD3100 (5 mg/g sq) and 40 x 106 donor marrow cells (from ROSA26 mice) were transplanted (via tail vein infusion) 6 h later, 2 h later, or both 2 and 6 h later (n=3 mice per condition). Control animals received donor cells but no AMD3100. Donor cell engraftment was assayed at 3m (6 h later group) and 2m (other groups, 3 m data are pending) and was significantly higher in the experimental animals than control. Engraftment was 3.0±1.6, 6.5±4.9, 6.5±3.6 (SD)%, respectively, in the AMD3100-treated mice, and 0.6±0.9, 0.6±0.8 and 2.0±1.9% in the concurrent control studies (all p values = 0.02). Confirmatory experiments in BalbC mice (where higher engraftment rates are anticipated in control studies (Blood 98:1246,2001)) are underway. Our data argue that the number of available niches determine the number of HSC that engraft. As importantly, mobilization with AMD3100 could provide a non-toxic preparative approach to HSC transplantation for genetic (and other nonmalignant) disorders.

Cyclophosphamide/granulocyte colony-stimulating factor induces hematopoietic stem cells to proliferate prior to mobilization.

We isolated hematopoietic stem cells (HSC) from mice treated with cyclophosphamide (CY) and granulocyte colony-stimulating factor (G-CSF). All mobilized multipotent progenitor activity was contained in two populations: Thy-1(lo) Sca-1+ Lin- Mac-1- CD4- c-kit+ long-term reconstituting progenitors and Thy-1(lo) Sca-1+ Lin- Mac-1(lo) CD4- transiently reconstituting progenitors. CY/G-CSF treatment drove both long-term and transient multipotent progenitors into cycle, leading to a more than 12-fold expansion in the number of long-term self-renewing HSC prior to mobilization. After CY and 2 days of G-CSF treatment the number of bone marrow HSC began to decline and the number of blood and splenic HSC increased. HSC continued to proliferate in the bone marrow and spleen through 8 days of G-CSF treatment, but HSC released into the blood tended to be in G0/G1 phase. Mobilized multipotent progenitors isolated from the spleen were less efficient than normal bone marrow multipotent progenitors in engrafting irradiated mice but did not differ in colony forming unit-spleen (CFU-S) activity or single cell in vitro assays of primitive progenitor activity. The data suggest that mobilized HSC isolated from the spleen are less efficient at homing to and engrafting the bone marrow of irradiated recipient mice.