Migration Of Hematopoietic Stem Cells Research Articles

BIGH3 modulates adhesion and migration of hematopoietic stem and progenitor cells

Cell adhesion and migration are important determinants of homing and development of hematopoietic stem and progenitor cells (HSPCs) in bone marrow (BM) niches. The extracellular matrix protein transforming growth factor-β (TGF-β) inducible gene H3 (BIGH3) is involved in adhesion and migration, although the effect of BIGH3 is highly cell type-dependent. BIGH3 is abundantly expressed by mesenchymal stromal cells, while its expression in HSPCs is relatively low unless induced by certain BM stressors. Here, we set out to determine how BIGH3 modulates HSPC adhesion and migration. We show that primary HSPCs adhere to BIGH3-coated substrates, which is, in part, integrin-dependent. Overexpression of BIGH3 in HSPCs and HL60 cells reduced the adhesion to the substrate fibronectin in adhesion assays, which was even more profound in electrical cell-substrate impedance sensing (ECIS) assays. Accordingly, the CXCL12 induced migration over fibronectin-coated surface was reduced in BIGH3-expressing HSPCs. The integrin expression profile of HSPCs was not altered upon BIGH3 expression. Although expression of BIGH3 did not alter actin polymerization in response to CXCL12, it inhibited the PMA-induced activation of the small GTPase RAC1 as well as the phosphorylation and activation of extracellular-regulated kinases (ERKs). Reduced activation of ERK and RAC1 may be responsible for the inhibition of cell adhesion and migration by BIGH3 in HSPCs. Induced BIGH3 expression upon BM stress may contribute to the regulation of BM homeostasis.

Dynamic Cross Talk between S1P and CXCL12 Regulates Hematopoietic Stem Cells Migration, Development and Bone Remodeling

Hematopoietic stem cells (HSCs) are mostly retained in a quiescent non-motile mode in their bone marrow (BM) niches, shifting to a migratory cycling and differentiating state to replenish the blood with mature leukocytes on demand. The balance between the major chemo-attractants CXCL12, predominantly in the BM, and S1P, mainly in the blood, dynamically regulates HSC recruitment to the circulation versus their retention in the BM. During alarm situations, stress-signals induce a decrease in CXCL12 levels in the BM, while S1P levels are rapidly and transiently increased in the circulation, thus favoring mobilization of stem cells as part of host defense and repair mechanisms. Myeloid cytokines, including G-CSF, up-regulate S1P signaling in the BM via the PI3K pathway. Induced CXCL12 secretion from stromal cells via reactive oxygen species (ROS) generation and increased S1P1 expression and ROS signaling in HSCs, all facilitate mobilization. Bone turnover is also modulated by both CXCL12 and S1P, regulating the dynamic BM stromal microenvironment, osteoclasts and stem cell niches which all functionally express CXCL12 and S1P receptors. Overall, CXCL12 and S1P levels in the BM and circulation are synchronized to mutually control HSC motility, leukocyte production and osteoclast/osteoblast bone turnover during homeostasis and stress situations.

Transcriptome Analysis Identifies Regulators of Hematopoietic Stem and Progenitor Cells

SummaryHematopoietic stem cells (HSCs) maintain blood homeostasis and are the functional units of bone marrow transplantation. To improve the molecular understanding of HSCs and their proximal progenitors, we performed transcriptome analysis within the context of the ImmGen Consortium data set. Gene sets that define steady-state and mobilized HSCs, as well as hematopoietic stem and progenitor cells (HSPCs), were determined. Genes involved in transcriptional regulation, including a group of putative transcriptional repressors, were identified in multipotent progenitors and HSCs. Proximal promoter analyses combined with ImmGen module analysis identified candidate regulators of HSCs. Enforced expression of one predicted regulator, Hlf, in diverse HSPC subsets led to extensive self-renewal activity ex vivo. These analyses reveal unique insights into the mechanisms that control the core properties of HSPCs.

Quantification of stem cell / niche interactions by coupling in vivo imaging and in silico simulation

For decades the bone marrow has been recognized as the home of hematopoietic stem cells (HSCs). Their regulation has been studied extensively, in particular regarding single molecules and pathways, but a comprehensive and dynamic picture is still elusive. This holds especially true for spatial organization. Recent advances in imaging techniques now allow acquisition of data on position and migration of HSCs in vivo. However, those techniques are limited by a relatively small observation volume and time span due to experimental limitations.

Hypoxia and Metabolic Properties of Hematopoietic Stem Cells

The effect of redox signaling on hematopoietic stem cell (HSC) function is not clearly understood. A growing body of evidence suggests that adult HSCs reside in the hypoxic bone marrow microenvironment or niche during homeostasis. It was recently shown that primitive HSCs in the bone marrow prefer to utilize anaerobic glycolysis to meet their energy demands and have lower rates of oxygen consumption and lower ATP levels. Hypoxia-inducible factor-α (Hif-1α) is a master regulator of cellular metabolism. With hundreds of downstream target genes and crosstalk with other signaling pathways, it regulates various aspects of metabolism from the oxidative stress response to glycolysis and mitochondrial respiration. Hif-1α is highly expressed in HSCs, where it regulates their function and metabolic phenotype. However, the regulation of Hif-1α in HSCs is not entirely understood. The homeobox transcription factor myeloid ecotropic viral integration site 1 (Meis1) is expressed in the most primitive HSCs populations, and it is required for primitive hematopoiesis. Recent reports suggest that Meis1 is required for normal adult HSC function by regulating the metabolism and redox state of HSCs transcriptionally through Hif-1α and Hif-2α. Given the profound effect of redox status on HSC function, it is critical to fully characterize the intrinsic, and microenvironment-related mechanisms of metabolic and redox regulation in HSCs. Future studies will be needed to elucidate the link between HSC metabolism and HSC fates, including quiescence, self-renewal, differentiation, apoptosis, and migration.

CXCL14 is a natural inhibitor of the CXCL12–CXCR4 signaling axis

Activation of the CXCL12–CXCR4 pathway is crucial for the migration of hematopoietic stem cells, various immune cells, and malignant tumor cells. Here, we show that another CXC chemokine, CXCL14, specifically binds to CXCR4 with high affinity and inhibits the CXCL12-mediated chemotaxis of human leukemia-derived cell lines and CD34+ hematopoietic progenitor cells. Thus, CXCL14 functions as a natural inhibitor of CXCL12. Our observations suggest that CXCL14 represents, along with CXCR7, molecules that co-evolved with the CXCL12–CXCR4 axis to modulate important physiological processes in development, stem cell maintenance, and immune responses. Structured summary of protein interactionsCXCR4physically interacts with CXCL14anti bait coimmunoprecipitation by (View interaction).

The tissue inhibitor of metalloproteinases-1 improves migration and adhesion of hematopoietic stem and progenitor cells

Homing and engraftment of hematopoietic stem and progenitor cells (HSPCs) during bone marrow transplantation are critically dependent on integrins such as β1-integrin. In the present study, we show that β1-integrin and the tetraspanin CD63 form a cell surface receptor complex for the soluble serum protein tissue inhibitor of metalloproteinases-1 (TIMP-1) on human CD34⁺ HSPCs. Through binding to this receptor complex, TIMP-1 activates β1-integrin, increases adhesion and migration of human CD34⁺ cells, and protects these cells from induced apoptosis. TIMP-1 stimulation in murine bone marrow mononuclear cells also promotes migration and adhesion; this is associated with augmented homing of murine mononuclear cells and of murine LSK⁺ cells during bone marrow transplantation. These results not only indicate that TIMP-1 is conducive to HSPC homing; they also identify CD63 and β1-integrin as a TIMP-1 receptor complex on HSPCs.

The histone demethylase UTX regulates stem cell migration and hematopoiesis

AbstractRegulated migration of hematopoietic stem cells is fundamental for hematopoiesis. The molecular mechanisms underlying stem cell trafficking are poorly defined. Based on a short hairpin RNA library and stromal cell-derived factor-1 (SDF-1) migration screening assay, we identified the histone 3 lysine 27 demethylase UTX (Kdm6a) as a novel regulator for hematopoietic cell migration. Using hematopoietic stem and progenitor cells from our conditional UTX knockout (KO) mice, we were able to confirm the regulatory function of UTX on cell migration. Moreover, adult female conditional UTX KO mice displayed myelodysplasia and splenic erythropoiesis, whereas UTX KO males showed no phenotype. During development, all UTX KO female and a portion of UTX KO male embryos developed a cardiac defect, cranioschisis, and died in utero. Therefore, UTY, the male homolog of UTX, can compensate for UTX in adults and partially during development. Additionally, we found that UTX knockdown in zebrafish significantly impairs SDF-1/CXCR4–dependent migration of primordial germ cells. Our data suggest that UTX is a critical regulator for stem cell migration and hematopoiesis.

Shear-Optimized Platelet-Like-Particles from High Ploidy MKS: From Segregation to Composition and Activation

Abstract Abstract 3456 Megakaryocytes (MKs) are rare cells that generate about 1010 platelets every day, which are necessary for vascular homeostasis via clot formation and contraction. Understanding MK differentiation and platelet production is of broad clinical importance and extends to a need to augment platelet numbers in patients. Our group has demonstrated that reversible but sustained inhibition of non-muscle myosin-II (NMM-II) increases MK polyploidization, proplatelet formation, and membrane flexibility, thereby increasing platelet generation (Shin, Spinler, et al., PNAS, 2011; 108:11458–63). The terminal steps of hematopoiesis involve the coordinated maturation and migration of multipotent hematopoietic stem cells (HSCs) from the bone marrow to the perivascular niche. Bulky, polyploid MKs do not easily transmigrate into blood, but do extend tubular membrane ‘proplatelet' projections into the bloodstream. Visualization by others has shown that blood shear stress fragments the projections into circulating platelets, motivating a controlled study of shear effects on MKs in vitro. To recapitulate this environment in vitro, a cone and plate rheometer is used to impart physiologically relevant shear stress on MEG01s, a human MK cell line. These experiments reveal that platelet-like-particle (PLP) generation is sensitive to both biomechanical and pharmacological factors namely blebbistatin inhibition of NMM-II. We demonstrate that shear stress reduces phosphor-deactivation of NMM-II heavy chain at Ser1943 to approximately 30% of the unsheared level, which indicates a restoration of NMM-II activity necessary for proper platelet function. Stimulation of rheometer generated PLP cultures with collagen-I showed aggregation and phosphatidylserine exposure (with Annexin-V binding in the presence of Ca2+). These data demonstrates that PLPs generated in this system retain some degree of functionality such that MKs exposed to shear stress and blebbistatin result in approximately 6.5 fold more PLPs than untreated MK cultures. Other groups have shown the transport and assembly of platelet organelles to be microtubule dependent and occur de novo along proplatelets (Italiano, et al, Blood, 2005; 106:4066–75). To assess partitioning and segregation of proteins from MKs in sheared membranes, we used fluorescence-imaged micro-deformation (FIMD) to monitor CD41 and NMM-IIA during micropipette aspiration. Studies of erythrocytes had already shown a rich variety of membrane component responses to membrane distension (Discher, et al, Science, 1994; 266:1032–5), but the methods have not yet been applied to MKs. Antibody labeling of MK surface CD41 shows a homogeneous intensity along the aspirated projection of membrane, but cell body shows approximately 3 fold higher intensity, suggesting an excess of CD41 in the MK. Pre-treatment with blebbistatin increases fragmentation frequency, and these fragments show a similar trend with CD41 expression. Nucleofection was used to introduce either GFP tagged WT NMM-IIA or phosphomemetic, myosin deactivating, GFP tagged NMM-IIA S1943D to assess whether pSer impacts partitioning of this cytoskeletal protein that is abundant in platelets. Both WT and S1943D NMM-IIA are seen in the aspirated cell projection, but WT NMM-IIA clearly accumulates at the leading edge of the aspirated projection and at sites of membrane fission and fragmentation, whereas S1943D remains uniformly dispersed. These findings thus underscore the central role that NMM-II heavy chain phosphorylation, and thus activity, in proplatelet formation and platelet fragmentation. Disclosures: No relevant conflicts of interest to declare.

Active RHOA favors retention of human hematopoietic stem/progenitor cells in their niche

BackgroundHematopoietic stem/progenitor cells (HSPCs) maintain the hematopoietic system by balancing their self-renewal and differentiation events. Hematopoietic stem cells also migrate to various sites and interact with their specific microenvironment to maintain the integrity of the system. Rho GTPases have been found to control the migration of hematopoietic cells and other cell types. Although the role of RAC1, RAC2 and CDC42 has been studied, the role of RHOA in human hematopoietic stem cells is unclear.ResultsBy utilizing constitutively active and dominant negative RHOA, we show that RHOA negatively regulates both in vitro and in vivo migration and dominant negative RHOA significantly increased the migration potential of human HSC/HPCs. Active RHOA expression favors the retention of hematopoietic stem/progenitor cells in the niche rather than migration and was found to lock the cells in the G0 cell cycle phase thereby affecting their long-term self-renewal potential.ConclusionThe current study demonstrates that down-regulation of RHOA might be used to facilitate the migration and homing of hematopoietic stem cells without affecting their long-term repopulating ability. This might be of interest especially for increasing the homing of ex vivo expanded HSPC.

Regulatory systems in bone marrow for hematopoietic stem/progenitor cells mobilization and homing.

Regulation of hematopoietic stem cell release, migration, and homing from the bone marrow (BM) and of the mobilization pathway involves a complex interaction among adhesion molecules, cytokines, proteolytic enzymes, stromal cells, and hematopoietic cells. The identification of new mechanisms that regulate the trafficking of hematopoietic stem/progenitor cells (HSPCs) cells has important implications, not only for hematopoietic transplantation but also for cell therapies in regenerative medicine for patients with acute myocardial infarction, spinal cord injury, and stroke, among others. This paper reviews the regulation mechanisms underlying the homing and mobilization of BM hematopoietic stem/progenitor cells, investigating the following issues: (a) the role of different factors, such as stromal cell derived factor-1 (SDF-1), granulocyte colony-stimulating factor (G-CSF), and vascular cell adhesion molecule-1 (VCAM-1), among other ligands; (b) the stem cell count in peripheral blood and BM and influential factors; (c) the therapeutic utilization of this phenomenon in lesions in different tissues, examining the agents involved in HSPCs mobilization, such as the different forms of G-CSF, plerixafor, and natalizumab; and (d) the effects of this mobilization on BM-derived stem/progenitor cells in clinical trials of patients with different diseases.

Role of the Ether-a-gò-gò-Related Gene 1B Isoform in Hematopoiesis

AbstractAbstract 1222Normal hematopoiesis is characterized by the tuned regulation of self renewal, proliferation, differentiation and migration of hematopoietic stem cells and HSC-derived multipotent and lineage-committed hemopoietic progenitor cells. This regulation is a complex process, which requires several levels of control provided by the activity of numerous membrane receptors and soluble proteins, which mediate the communication among hematopoietic cells, and between the cells and the microenvironment. In this contest, ion channels must be mentioned. Indeed, besides their canonical role in cell excitability, they can also modulate different cellular functions, such as proliferation, apoptosis and differentiation, in both excitable and non excitable cells. This role is also relevant in hematopoietic cells, where ion channels have a clear role in different functions of fully differentiated cells (Int Rev Cell Mol Biol. 2010;279:135–190).On these bases, we analyzed the role of ether a gò-gò-related gene 1 (ERG1) channels in normal hematopoiesis. In particular, we performed lack of function studies using a murine ERG1 knock out (KO) model (in SV129 strain). Since mice with a general and complete KO of the whole ERG1 gene die during early development, we analyzed mice with a selective deletion of the ERG1B isoform (ERG1B−/− mice, Mol Cell Biol. 1003;23(6):1856–1862), which is the ERG1 isoform mostly expressed in leukemic blasts (Blood. 2007;110(4):1238–1250). Such mice are viable and do not show any life threatening physical or behavioral abnormalities. First, we verified ERG1 transcripts expression in wild type SV129 mice (WT): both transcripts were expressed in spleen and thymus with higher values for the ERG1A isoform; ERG1B isoform presented a good expression level in bone marrow (BM) especially in the Sca-1+ population. Consequently we performed experiments to evaluate the role of ERG1B in normal hematopoiesis. Young (0–3 months old) KO mice presented a reduced number of CFUs (colony forming units) in the BM. CFUs levels were restored in adult mice. BM of KO mice showed hypocellularity and an increased number of megakaryocytes intriguingly associated with a reduction of erythrocytes (Ter119+). As evidenced by the histological analysis, splenomegaly of KO mice could be traced back to a great amount of mature red blood cells, filling the interfollicular space of the red pulp and subcapsulary space. Such splenic congestion in ERG1B−/− mice is accompanied by a relative decrease in the number of megakaryocytes, as well as by a reduced capacity to develop CFUs. On the whole, these data are suggestive of a failure of spleen hemopoiesis, with a concomitant red cell engulfment that lead to a putative erythropoiesis that occurs locally in the spleen maybe due to reprogrammation of hematopoietic cells of different lineage. To better characterize hematopoiesis in ERG1B−/− mouse model we performed two different stress tests: myelotoxicity and acute hemolytic anemia induction. Myelotoxicity was induced by single dose injection of cyclophosphamide (450 mg/Kg) in both WT and KO mice. From this induction we expected a decreased myelopoiesis, mostly affecting granulocytes and monocytes, followed by a rebound due to the capacity of the mice to undertake myelopoiesis. In KO model this compensation was absent suggesting a functional defect into the myeloid lineage correlated with ERG1B deletion. Finally, we induced acute anemia in mice testing their response to phenylhydrazine (PHZ, 60 mg/Kg). As expected, in WT mice, RBCs value rapidly declined followed by a compensatory erythropoiesis. In ERG1B−/− mice, we observed a reduced capacity to recover physiological RBCs values. Such results suggest that a functional defect occurred also into the erythroid lineage.On the whole, the present study provides evidence that the ERG1B isoform exerts a relevant role in hematopoiesis, driving the commitment and maturation of different hematopoietic cell populations. Disclosures:No relevant conflicts of interest to declare.

Vav1 Deficiency Differentially Affects Engraftment of Fetal Hematopoietic Organs During Development Compared with Adult Marrow After Transplantation

Abstract 1212Rac GTPases function as molecular switches, which integrate extracellular signals that are critical for hematopoietic stem cell (HSC) engraftment and bone marrow repopulation during transplantation. We have recently reported that loss of the Vav1 protein, a known hematopoietic-specific activator of Rac, drastically reduces engraftment of adult HSCs without compromising steady-state hematopoiesis in adult mice (Sanchez-Aguilera et al., PNAS 2011). This suggests that HSC migration during ontogeny is unaffected by Vav1 loss and implies that GTPase regulation of HSC migration is distinctly different during embryonic migration versus (vs.) migration required during transplant-mediated engraftment. To verify this, we have examined the role of Vav1 in fetal hematopoietic cells. In the developing embryo, Vav1 expression is first detected at E11.5 and increases to E14.5 (Bustelo et al., Cell Growth Differ 1993). At this developmental age, fetal liver (FL) is the major hematopoietic organ in the embryo. We found no difference between Vav1−/− and WT animals in the number of immunophenotypically defined HSCs (LSK SLAM or c-Kit+CD34+) in the FL isolated from E13.5 embryo (4.4 +/− 0.6% LSK-SLAM in the WT group vs. 7.6 +/− 1.4 in the Vav1−/−group). Vav1−/− FL cells also formed normal numbers of hematopoietic progenitor colonies in methylcellulose (42.01 +/−3.2 in the WT group vs. 37.63 +/− 2.5 in the Vav1−/−group). We then compared the number of LSK SLAM cells in the BM of Vav1−/− newborn mice to WT, as well as their ability to form colonies in methylcellulose, and did not detect any difference between the two genotypes. Together, these data suggest that during hematopoietic development, the initial colonization of the FL and subsequent transition to BM hematopoiesis are not affected by deletion of Vav1. To determine whether Vav1 is required for engraftment of FL cells after transplantation, we performed competitive transplantation experiments. We found that Vav1−/− E13.5 FL cells failed to provide long term engraftment to lethally irradiated syngeneic recipients, as measured by peripheral blood (PB) chimerism at 4, 8 and 16 weeks after transplantation (an average of 0.08%, 0.42% and 0.12% donor-derived CD45.2 positive cells in the PB of Vav mice at the three time points analyzed vs. 42.1%, 49.5% and 66.35% in the WT group; p<0.0001, p=0.0049, p=0.0016, respectively). In addition, Vav1−/− FL cells failed to contribute to PB reconstitution in newborn syngeneic recipients that were sublethally irradiated or born from pregnant mothers conditioned with busulfan treatment. These data confirm the differential requirement for Vav1 during fetal physiological organ engraftment compared to HSC transplant. Next, we sought to determine the mechanisms underlying the lack of engraftment of Vav1−/− fetal cells during transplantation. Given that the Vav1-Rac axis has been shown to contribute to the activation of the JNK pathway (Crespo et al., Oncogene 1996), which is induced by pro-inflammatory cytokines (Kyaris et al., Nature 1994) produced during bone marrow ablation, we tested whether Vav1−/− cells were hypersensitive to pro-inflammatory cytokines. Vav1−/− cells produced normal numbers of progenitor-derived colonies in vitro in the presence of increasing doses of TNFα and IFNγ. Similarly, the lack of engraftment of Vav1−/−HSC was not rescued upon transplantation into W/Wv mice, which do not require any myelablation for effective engraftment of WT HSC. These results indicate that the lack of engraftment of Vav1 cells is not due to an increased sensitivity to pro-inflammatory cytokines.In conclusion, our data suggest that distinct mechanisms regulate the physiological migration of FL cells to the BM during development and their ability to reconstitute the hematopoietic system of recipients during transplantation. We believe that understanding the basis of these mechanisms is critical for transplant biology and regenerative medicine. Disclosures:Williams:bluebird bio: Consultancy; Wyeth:; Takara bio:.

Shear-Optimized Platelet-Like-Particles From High Ploidy Mks: From Segregation to Composition and Activation

Abstract 3456Megakaryocytes (MKs) are rare cells that generate about 1010 platelets every day, which are necessary for vascular homeostasis via clot formation and contraction. Understanding MK differentiation and platelet production is of broad clinical importance and extends to a need to augment platelet numbers in patients. Our group has demonstrated that reversible but sustained inhibition of non-muscle myosin-II (NMM-II) increases MK polyploidization, proplatelet formation, and membrane flexibility, thereby increasing platelet generation (Shin, Spinler, et al., PNAS, 2011; 108:11458–63). The terminal steps of hematopoiesis involve the coordinated maturation and migration of multipotent hematopoietic stem cells (HSCs) from the bone marrow to the perivascular niche. Bulky, polyploid MKs do not easily transmigrate into blood, but do extend tubular membrane ‘proplatelet' projections into the bloodstream. Visualization by others has shown that blood shear stress fragments the projections into circulating platelets, motivating a controlled study of shear effects on MKs in vitro.To recapitulate this environment in vitro, a cone and plate rheometer is used to impart physiologically relevant shear stress on MEG01s, a human MK cell line. These experiments reveal that platelet-like-particle (PLP) generation is sensitive to both biomechanical and pharmacological factors namely blebbistatin inhibition of NMM-II. We demonstrate that shear stress reduces phosphor-deactivation of NMM-II heavy chain at Ser1943 to approximately 30% of the unsheared level, which indicates a restoration of NMM-II activity necessary for proper platelet function. Stimulation of rheometer generated PLP cultures with collagen-I showed aggregation and phosphatidylserine exposure (with Annexin-V binding in the presence of Ca2+). These data demonstrates that PLPs generated in this system retain some degree of functionality such that MKs exposed to shear stress and blebbistatin result in approximately 6.5 fold more PLPs than untreated MK cultures.Other groups have shown the transport and assembly of platelet organelles to be microtubule dependent and occur de novo along proplatelets (Italiano, et al, Blood, 2005; 106:4066–75). To assess partitioning and segregation of proteins from MKs in sheared membranes, we used fluorescence-imaged micro-deformation (FIMD) to monitor CD41 and NMM-IIA during micropipette aspiration. Studies of erythrocytes had already shown a rich variety of membrane component responses to membrane distension (Discher, et al, Science, 1994; 266:1032–5), but the methods have not yet been applied to MKs. Antibody labeling of MK surface CD41 shows a homogeneous intensity along the aspirated projection of membrane, but cell body shows approximately 3 fold higher intensity, suggesting an excess of CD41 in the MK. Pre-treatment with blebbistatin increases fragmentation frequency, and these fragments show a similar trend with CD41 expression. Nucleofection was used to introduce either GFP tagged WT NMM-IIA or phosphomemetic, myosin deactivating, GFP tagged NMM-IIA S1943D to assess whether pSer impacts partitioning of this cytoskeletal protein that is abundant in platelets. Both WT and S1943D NMM-IIA are seen in the aspirated cell projection, but WT NMM-IIA clearly accumulates at the leading edge of the aspirated projection and at sites of membrane fission and fragmentation, whereas S1943D remains uniformly dispersed. These findings thus underscore the central role that NMM-II heavy chain phosphorylation, and thus activity, in proplatelet formation and platelet fragmentation. Disclosures:No relevant conflicts of interest to declare.

Sinusoidal Endothelial CD26 Cleavage of Neuropeptide Y Regulates Transendothelial Migration and Mobilization of Hematopoietic Stem and Progenitor Cells in Response to G-CSF.

Abstract 2347Hematopoietic stem and progenitor cell (HSPC) mobilization by G-CSF is reduced in CD26 knockout (−/−) mice or mice treated with compounds that inhibit CD26 enzyme activity. However, how CD26 regulates mobilization is not known. CD26 is a surface dipeptidyl peptidase expressed on hematopoietic and non-hematopoietic cells. The fact that CD26 is expressed on HSPC, has suggested that intrinsic HSPC CD26 is involved in their mobilization. We reasoned that if intrinsic HSPC CD26 is crucial for their mobilization, then CD26+ HSPC would mobilize more readily than CD26− HSPC. After G-CSF treatment, CD26+ SLAM LSK and LSK cells increased 2 and 3.7-fold in peripheral blood (PB) whereas CD26− SLAM LSK and LSK cells increased 3.9 and 8.7 fold respectively; with the ratio of CD26+/CD26− HSPC in the bone marrow (BM) remaining unchanged. In chimeric mice where hematopoietic cells were either wild-type or CD26−/− in origin and the stromal environment was wild-type, equivalent CFU-GM and LSK cells were mobilized by G-CSF. These studies suggest that CD26 expression on hematopoietic origin cells including HSPC is not required for the G-CSF induced mobilization of HSPC.The BM stromal environment includes both osteolineage cells and endothelial cells (EC). To explore whether osteolineage cell CD26 is involved in G-CSF mobilization, we quantitated CD45−CD119−CD31−Sca-1+ Alcam− cells enriched for mesenchymal progenitor cells (MPC) and CD45−CD119−CD31− Sca-1- Alcam+ cells enriched for osteoblasts (OB) in the BM of mice treated with G-CSF or G-CSF plus the CD26 inhibitor diprotin A. Total MPC and OB number as well as the retention factors SDF-1, SCF-1 and osteoprotegerin (OPG) were reduced equally in the BM of G-CSF and G-CSF plus diprotin A treated mice, suggesting that osteolineage cell CD26 is not involved in mobilization. Since endothelial cells (EC) express CD26 and HSPC resides proximal to sinusoidal endothelial cells (SEC), we evaluated if SEC CD26 has a role in G-CSF induced HSPC mobilization. G-CSF treatment increased CD26 expression on CD45- Ter119− Sca-1− VEGFR3+ SEC by 2-fold. Because mobilized HSPC must transmigrate across SEC to enter the PB, we determined if EC CD26 regulates HSPC transmigration. Treatment of confluent EC monolayers with G-CSF in vitro, increased transendothelial migration of both wild-type and CD26−/−HSPC, which was significantly reduced by diprotin A, supporting the hypothesis that enhanced expression of CD26 on BM SEC by G-CSF is involved in HSPC mobilization.Since SDF-1 is a BM retention factor for HSPC and CD26 can cleave SDF-1 in vitro, it is possible that SDF-1 degradation by CD26 plays a role in HSPC egress. However, direct evidence supporting proteolytic inactivation of SDF-1 by CD26 in response to G-CSF is not available. Using a novel bio-binding assay and mass spectroscopy, we found similar levels of active SDF-1 in the BM extracellular fluid and on SEC membranes from G-CSF treated wild-type and CD26−/− mice, suggesting that G-CSF mobilization is not linked to SDF-1degradation. We therefore searched protein sequence databases for known EC products containing a CD26 recognition site and implicated in HSPC trafficking. We found that the neurotransmitter neuropeptide Y (NPY), a sympathetic nerve and EC product contains an N-terminal Tyr-Pro sequence and can be cleaved to NPY3–36 by CD26. We hypothesized that if cleavage of NPY by EC CD26 is involved in transendothelial migration of HSPC, then reduced HSPC migration through EC monolayers in the presence of G-CSF plus diprotin A would be reversed in the presence of NPY3–36. As expected, NPY3–36 reversed the diprotin A mediated attenuation in HSPC migration through G-CSF treated EC. In vivo, the attenuated response to G-CSF mobilization in diprotin A treated or CD26−/− mice could be completely reversed by co-administration of NPY3–36, but not full length NPY. In G-CSF mobilized wild-type mice, we found that expression of gap junction proteins on BM SEC, including CD31 and VE-cadherin are reduced coincident with increased EC permeability, which is attenuated by co-administration of diptotin A and restored to normal in mice receiving G-CSF plus diprotin A and NPY3–36, but not full length NPY. In conclusion, we have found that CD26 regulates G-CSF induced mobilization that is independent of the SDF-1 paradigm; rather elevated SEC CD26 in response to G-CSF enhances transendothelial migration and mobilization of HSPC via cleavage of NPY. Disclosures:No relevant conflicts of interest to declare.

Microbial Impact on Hematologic Homeostasis

AbstractAbstract SCI-40During systemic infection and inflammation, immune effector cells are in high demand and are rapidly consumed at sites of need. While adaptive immune cells have high proliferative potential, innate mature immune cells are mostly postmitotic and need to be replenished from bone marrow hematopoietic stem and progenitor cells. Indeed, severe clinical infection, particularly infections challenging the innate immune response, lead to an increase in hematopoietic differentiation and throughput in bone marrow, involving subsequent differentiation stages from hematopoietic stem cells, multipotent progenitors, as well as early-lineage and late-lineage restricted hematopoietic progenitors. A fundamental question is how the increased need is sensed and translated in enhanced production and how adequate levels of response are guided. Recent research has shed light on conserved intracellular and extracellular pathogen recognition receptors, such as Toll-like receptors, that are expressed on nonhematopoietic and hematopoietic effector cells and cause activation upon ligation. This activation results in production of hematopoietic growth, survival, activation, and migration factors operating at site on effector cells, but also at remote primary hematopoietic sites to increase production upon need. Recent research by several groups, including ours, surprisingly revealed that conserved pattern-recognition receptors are also expressed on hematopoietic stem and progenitor cells in bone marrow, implying a direct effect of systemically available ligands on these cellular populations. Indeed, it has been demonstrated that, for example, ligation of Toll-like receptor 4 by its cognate agonist lipopolysaccharide can lead to divisional activation, proliferation, lineage-directed differentiation, and migration of hematopoietic stem and lineage-restricted progenitor cells, all aimed at efficient contribution to immune responses and rapid reestablishment of hematopoietic homeostasis. The relative contribution of pathogen sensing by hematopoietic and diverse nonhematopoietic cells to appropriate hematopoietic responses, as well as the subcellular translation of the signals, is the focus of ongoing research. Also to be discussed will be how chronic infectious and inflammatory processes, which are frequently associated with aging, might impinge on hematopoiesis, potentially fostering hematopoietic stem cell diseases as exhaustion or transformation. Disclosures:No relevant conflicts of interest to declare.

Role of N-Cadherin in the Regulation of Hematopoietic Stem Cells in the Fetal Liver and Bone Marrow

Abstract 1205The interaction of stem cells with their supportive microenvironmental niche is critical for sustaining stem cell pools in tissues over long periods of time. Cell-cell and cell-extracellular matrix interactions between hematopoietic stem cells (HSCs) and their niches contribute to the maintenance of stem cell properties. We previously demonstrated that N-cadherin mediated cell adhesion plays a critical role in the HSC engraftment and slow cell division of HSCs. Furthermore, in vitro culture of HSCs with bone-derived osteoblasts that expressed high levels of N-cadherin enhanced the LTR activity of HSCs. However, the expression and function of N-cadherin in HSCs is still controversial. A major problem is that there have been no specific anti-N-cadherin antibodies (Abs) that can be used for the detection of N-cadherin on the surface of living cells.To address this problem, we produced a new anti-N-cadherin Ab. For the production of anti-N-cadherin Abs, we used the phage display library and isolated recombinant Ab clones against mouse N-cadherin. After screening of the phage library and performing quality control ELISA with positive and negative control proteins, we found that three of the seven newly-developed Ab clones were suitable for FACS. FACS analysis with a new N-cadherin Ab showed that BM LSK cells expressed low levels of N-cadherin protein. Furthermore, we confirmed that the reactivity of the new N-cadherin Ab was significantly reduced in N-cadherin deficient LSK cells compared to the wild-type LSK cells. RT-PCR and Q-PCR analysis revealed significantly higher levels of N-cadherin mRNA in N-cadherin+ LSK cells compared with N-cadherin– LSK cells. Next, we performed BMT assays with adult BM-derived N-cadherin+ and N-cadherin– LSK cells isolated by using the new N-cadherin Ab, clone AbD13081, and found that N-cadherin+ LSK cells showed higher BM reconstitution compared with N-cadherin– cells. Furthermore, one of our N-cadherin Ab clones, AbD13077, has neutralizing activity and the use of this clone in cell sorting reduces the LTR activity of N-cadherin+ LSK cells. These data suggested that adult BM HSCs express N-cadherin.Next we examined the expression of N-cadherin in the fetal HSCs using a new N-cadherin Ab. We found that a large number (29.3 ± 2.6 %) of LSK cells in E12.5 fetal liver (FL) expressed N-cadherin. Interestingly, N-cadherin expression was drastically decreased in E15.5 and E18.5 FL LSK cells (13.2 ± 1.9 % in E15.5 and 16.5 ± 1.4 % in E18.5). Immunohistochemical staining revealed that N-cadherin+c-Kit+ cells/N-cadherin+EPCR+ hematopoietic cells adhered to Lyve-1+ endothelial cells in E12.5 FL. Consistent with FACS analysis, N-cadherin expression was decreased in E15.5 and E18.5 FL. In contrast, the expression of E-cadherin in hepatic cells was significantly upregulated in E15.5 and E18.5 FL. Next we analyzed the expression of the LT-HSC marker, EPCR in N-cadherin+ and N-cadherin− LSK cells. We found that EPCR+ cells were enriched in the N-cadherin+ LSK fraction in E12.5 FL, while there was no significant difference in the frequency of EPCR+ cells between N-cadherin+ and N-cadherin− LSK in E15.5 and E18.5 FL LSK cells.Finally, we performed the BMT assay with E12.5, E15.5, and E18.5 FL-derived N-cadherin+ and N-cadherin– LSK cells isolated by AbD13081. Similar to the BM N-cadherin+ LSK cells, E12.5 FL N-cadherin+ LSK cells showed higher LTR activity than N-cadherin– LSK cells. Interestingly, the advantage of LTR in N-cadherin+ LSK cells was decreased in E15.5 and E18.5 FL compared to E12.5 N-cadherin+ LSK cells, although the reconstitution of the N-cadherin+ fraction was higher than N-cadherin– fraction.Altogether, these data suggest that N-cadherin is highly expressed in E12.5 FL HSCs and plays an important role in the HSC-niche interaction for the maintenance of HSC activity. We speculated that the decrease of N-cadherin in HSC and FL cells during FL development might contribute to the migration of HSCs from FL to BM. Disclosures:No relevant conflicts of interest to declare.

Identification and Phenotypic Characterization of a Subpopulation of Acute Myelogenous Leukemia (AML) Cells with Increased Plastic Adherence.

Abstract 2556The high incidence of relapse in acute myelogenous leukemia (AML) patients has been attributed to the existence of a small population of leukemic stem cells (LSC) that current therapies are unable to eradicate. LSC, in similarity to normal hematopoietic stem cells (HSC), are able to engraft, self-renew, and interact with cells within the bone marrow (BM) niche. During leukemogenesis, changes within the BM microenvironment promote LSC survival and expansion, and shelter leukemic cells from chemotherapy. Therefore, displacing these cells from the BM niches prior to chemotherapy may decrease drug resistance and prevent relapse of the disease. The binding of α4β1 integrins (VLA-4 or CD49d/CD29) to the stromal extracellular matrix and cell surface ligands is a key component in homing and trafficking, specifically in the migration of HSC beneath marrow stromal cells, and VLA-4 has been shown to be crucial for the persistence of minimal residual disease in AML. KG1a is a differentiation-resistant AML cell line containing a very primitive population of CD34+CD38- cells. Upon culture, the majority of KG1a cells remain in suspension, while a small percentage adheres to the tissue culture plastic (Adh-KG1a). Here, we hypothesized that characterization of adhesion molecules that were either unique or significantly altered in this subset could lead to identification of putative therapeutic targets for dislodging AML cells from microenvironmental niches. KG1a and Adh-KG1a populations were evaluated by flow-cytometry for the presence of CD49d and CD29.The FACSort results were then analyzed using FlowJo7.6 software. No statistically significant (p>0.05) differences were found in the percentage of Adh-KG1a and KG1a that expressed CD49d (91.6±3% vs. 86.2±5%) or CD29 (95.0±2% vs. 91.8±1.8%). However, we found that there is a small and unique population of adherent CD29+ cells, with lower fluorescent intensity (MFI=326), that is not present in the non-adherent population (MFI=435), suggesting that this low level of surface expression is unique to the adherent fraction. Analysis of CD11a, CD44, CD18, CD106, CD105, CD34, CD107 and CD38 showed that none of these molecules were expressed at significantly different levels between KG1a and the Adh-KG1a fraction. Addition of anti-CD44 or anti-CD29 antibodies to the cells in culture did not result in decreased numbers of Adh-KG1a cells/flask, but resulted in induction of heterotypic aggregation of Adh-KG1a. Cell cycle analysis did not show significant differences between the 2 populations. However, analysis of CD54 expression demonstrated that 12.2% more of the Adh-KG1a were positive for CD54 than the non-adherent cells. Furthermore, characterization of gangliosides on KG1a and Adh-KG1a showed that the non-adherent fraction contained significantly more GM3 than the Adh-KG1a. Given prior reports showing that: 1) high levels of VLA-4 (CD49d/CD29) are associated with better prognosis in AML; 2) high levels of CD54 correlate with low relapse-free survival probability in AML; and 3) ganglioside composition has been shown to exert a pronounced effect on drug-resistance/sensitivity in AML, our findings of an adherent population of primitive hematopoietic stem/progenitor cells within KG1a that are CD29dim, express high levels of CD54, and contain lower levels of GM3, suggest that this unique subpopulation may play an important role in relapse in AML, and could represent a target for novel therapeutics that could better eradicate the elusive LSC and promise an improved disease-free survival rate in AML. Disclosures:No relevant conflicts of interest to declare.

The Contact with MDS Endothelial Cells Alters the Pattern of Lineage-Specific Gene Expression During Normal Hematopoietic Differentiation

AbstractAbstract 1718In the embryo, hematopoiesis and angiogenesis occur concurrently, since endothelial and hematopoietic cells derive from a common cell precursor, the hemangioblast. In the post natal life, the bone marrow (BM) vascular niche has an important role in supporting hematopoietic stem cells (HSC) proliferation and differentiation. Moreover, during the processes of homing and mobilization, BM vascular niche regulates HSC migration. In BM failure, these functions are promoted by vascular niches located outside of the BM, such as in the spleen, thus confirming that endothelial cells are relevant to the normal hematopoietic differentiation. The dominant feature of myelodyspslatic syndromes (MDS) is the impaired capacity of HSC to give rise to a terminally differentiated normal cell population in one or more lineages. We hypothesized that in MDS patients the vascular niches within and eventually outside of the BM might be altered and that they do not support adequately the physiologic hematopoietic differentiation, thus contributing to the pathogenesis of the disease. To investigate this hypothesis, we isolated and expanded endothelial colony forming cells (ECFCs) (Ingram et al. Blood. 2004;104: 2752) from the peripheral blood of patients with MDS (RCMD) and we used ECFCs as a surrogate of the vascular niche to differentiate hematopoietic cells. Normal CD34+ were seeded over a layer of endothelial cells and cultured for 8 days in different cytokine- containing media able to induce selective granulocytic-macrophage, erythroid or megakaryocyte differentiation. At baseline and after 5 and 8 days of co-culture, hematopoietic cells were recovered by gentle repeated washings, and then they were analyzed with RT-PCR for the expression of lineage specific genes. For each lineage, two genes, mainly involved either in the early or in the late phase of the differentiation, respectively, were analyzed. Basically, we found that co-culturing CD34+ cells in contact with a normal endothelial layer the gene expression observed in the corresponding no-layer cultures was amplified. This effect was strictly dependent on the physical contact between hematopoietic and endothelial cells, since it was abolished when cells were grown separated from a porous membrane. We observed that in co-cultures with normal hematopoietic cells and MDS ECFCs, the modulatory effect exerted by the endothelial layer was partially or completely lost. For example, CD34+ cells undergoing granulocyte differentiation showed a progressive decline of PU.1 gene expression, followed by the rise of the expression of MPO gene; these effects were much more evident if cells were cultured over normal ECFCs. In contrast, this pattern of gene expression was deeply perturbed when normal CD34+ cells were cultured with MDS ECFCs: actually, at day 7, the PU.1 RNA level was six folds higher and the MPO RNA level was three folds lower than that observed in co-cultures with normal ECFCs. Similar figures were observed for RUNX1 and GP1b gene expression in cells undergoing megakaryocyte differentiation. Moreover, CD34+ cells undergoing erythroid differentiation on normal ECFCs exhibited the progressive up-regulation of FOG-1 and of transferrin receptor (TFR) gene expression: interestingly, the co-culture with MDS ECFCs resulted in a lower expression of FOG -1 gene but also in a surprisingly higher expression of the TFR gene. Overall, these data suggest that in MDS does exist an impairment of talking between endothelial and hematopoietic cells. To understand which molecular pathways are involved could represent the basis for a novel therapy approach. Disclosures:No relevant conflicts of interest to declare.

Hematopoietic stem cell mobilization into the peripheral circulation in patients with chronic liver diseases

The present study was aimed to investigate and compare the kinetics of bone marrow-derived hematopoietic stem cells (BMHSC) migration in the peripheral blood and liver in response to liver injury in patients with chronic liver disease (CLD). In all, 45 CLD patients staged with Child-Pugh A, B and C and 15 healthy participants were evaluated for the concentration of circulating BMHSC by a flow cytometric analysis of CD133(+) /CD34(+) cells. In addition, homing BMHSC and hepatic progenitors were assessed by the immunohistochemical detection of CD133(+) and OV6(+) cells in liver biopsy specimens from Child-Pugh A and B patients. No significant difference in the percentage of circulating CD133(+) /CD34(+) cells was observed among all groups of patients. In liver tissues, OV6(+) cells increased significantly in Child-Pugh B cases (P < 0.05), while CD133(+) cells were distributed sparsely in the periportal region in Child-Pugh A and B patients. OV6(+) cells were significantly correlated with CD34(+) cells but not with CD133(+) cells in Child-Pugh A and B patients (P < 0.01 and P < 0.05, respectively). Various degrees of severity in CLD neither evoked the mobilization of BMHSC into the circulation nor triggered their homing into liver tissue, thus excluding extrahepatic stem cell-mediated repair. The recovery process seems to be dependent on proliferating endogenous liver progenitors (OV6(+) cells).