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

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1222-1222
Author(s):  
Serena Pillozzi ◽  
Marika Masselli ◽  
Marinella Veltroni ◽  
Emanuele De Lorenzo ◽  
Antonella Fiore ◽  
...  
Keyword(s):  
Ion Channels ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Membrane Receptors ◽  
Stress Tests ◽  
Related Gene ◽  
Hematopoietic Stem ◽  
Knock Out ◽  
Functional Defect

Abstract Abstract 1222 Normal 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.

Endothelial Cells Are Essential to Sense Lipopolysaccharide in a MYD88-Dependent Manner and to Subsequently Induce Emergency Myelopoiesis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 641-641
Author(s):  
Steffen Boettcher ◽  
Rahel Gerosa ◽  
Ramin Radpour ◽  
Markus G. Manz
Keyword(s):  
Endothelial Cells ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Hematopoietic Cells ◽  
Clinical Signs ◽  
Dependent Manner ◽  
Perivascular Cells ◽  
Hematopoietic Stem ◽  
Emergency Myelopoiesis

Abstract Abstract 641 Severe systemic infections evoke a number of characteristic clinical signs such as fever, neutrophilia and the appearance of immature myeloid precursors in the circulation (left-shift). This reflects a well-regulated hematopoietic response program to enhance myeloid cell output during times of increased hematopoietic demand, a condition which is referred to as 'emergency myelopoiesis'. Important molecular components of the emergency myelopoiesis cascade, such as cytokines and transcription factors involved, have been elucidated. However, the initial steps of emergency myelopoiesis involving pathogen recognition and translation into accelerated bone marrow (BM) myelopoiesis have only been inferred from findings on Toll-like receptor (TLR) expression on immature hematopoietic stem and progenitor cells (HSPCs) as well as on mature hematopoietic cells (e.g. macrophages). Accordingly, it has been assumed that both immature as well as mature hematopoietic cells are involved in sensing infection and inducing emergency myelopoiesis directly and indirectly, respectively. Surprisingly, by generating reciprocal BM chimeric animals mice with TLR4−/− hematopoiesis on a wild-type (WT) nonhematopoietic background (TLR4−/−→WT mice) and WT hematopoiesis on a TLR4−/− nonhematopoietic background (WT→TLR4−/−mice), we demonstrated that LPS-Induced emergency myelopoiesis depends on TLR4-expressing nonhematopoietic cells (Boettcher et al., J Immunol. 2012 Jun 15;188(12):5824–8.). However, the precise identity and localization of the nonhematopoietic cell type crucial for sensing gramnegative infection-derived lipopolysaccharide (LPS) has remained elusive to date. We now have addressed this fundamental question using BM transplantation experiments and Cre-loxP recombination technology. BM chimeric mice with a myeloid differentiation primary response gene 88 (Myd88)-deficiency in the hematopoietic lineage (MYD88−/−→WT mice) showed a normal LPS response indistinguishable to control (WT→WT) mice, while knocked out Myd88 within the nonhematopoietic compartment (WT→MYD88−/− mice) led to a non-responsiveness towards LPS similar to controls (Myd88−/−→Myd88−/− mice). These results are in line with our earlier data, thus confirming the critical role of the TLR4/MYD88 pathway in nonhematopoietic cells for the induction of emergency myelopoiesis. In order to specifically delete TLR-MyYD88-downstream signaling in various nonhematopoietic cells including BM Nestin+ mesenchymal stem cells (MSCs) and their progeny, perivascular cells, endothelial cells, and hepatocytes, we generated Nes-Cre;Myd88fl/fl, Pdgfrb-Cre;Myd88fl/fl, Tek-Cre;Myd88fl/fl, and Alb-Cre;Myd88fl/fl mice, respectively. We observed a normal increase in the frequency of BM CD11b+Gr-1low immature myeloid precursors accompanied by a decrease of BM CD11b+Gr-1high mature myeloid cells upon LPS stimulation characteristic for efficient emergency myelopoiesis in Nes-Cre;Myd88fl/fl, Pdgfrb-Cre;Myd88fl/fl, and Alb-Cre;Myd88fl/fl mice as compared to control mice. Furthermore, we measured highly-elevated plasma G-CSF levels in these mouse strains upon LPS injection. Hence, intact TLR signaling in mesenchymal stromal cells incl. Nestin+ MSCs, perivascular cells as well as hepatocytes is dispensable for induction of emergency myelopoiesis. Strikingly, Tek-Cre;Myd88fl/fl mice were completely non-responsive towards LPS stimulation as assessed by the above-mentioned parameters. Our results thus demonstrate a fundamental and unanticipated role of the endothelium for sensing of systemically spread pathogens and subsequent stimulation of BM emergency myelopoiesis. Disclosures: No relevant conflicts of interest to declare.

The Role Of tet2 DNA Methylcytosine Dioxygenase In Zebrafish Early Hematopoiesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1204-1204
Author(s):  
Huan-Chau Lin ◽  
Ken-Hong Lim ◽  
Yi-Hao Chiang ◽  
Wei-Ting Wang ◽  
Ching-Sung Lin ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Mendelian Inheritance ◽  
Stem Cell Markers ◽  
Loss Of Function ◽  
Transcription Activator ◽  
Hematopoietic Stem ◽  
Mature Adult ◽  
Knock Out ◽  
Knock Down

Abstract Loss-of-function mutations in Ten-Eleven-Translocation 2 (TET2) gene have been identified in various human myeloid and lymphoid malignancies. Recently, the TET gene family (TET1, TET2, and TET3) was found to function as DNA methylcytosine dioxygenase that is able to oxidize 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC). In Tet2-deficient mouse models, Tet2 has been shown to play an important role in regulating self-renewal and differentiation of hematopoietic stem cells. These Tet2-deficient mice would gradually develop a chronic myeloid neoplasm resembling human chronic myelomonocytic leukemia suggesting that TET2 may function as a tumor suppressor. In the present study, we investigated the role of tet2 in zebrafish early hematopoiesis. During zebrafish early development, the expression of tet1, tet2, and tet3 by qRT-PCR can be detected mainly after the segmentation stage (26-somite), with fluctuated expression levels thereafter. Whole-mount in situ hybridization revealed that tet2 expression was strong over aorta-gonad-mesonephros region at 48 hours post-fertilization (hpf). Morpholino oligonucleotide (MO) knock-down of tet2 increased the expression of tet1, tet3, dnmt3aa, gata-1, alpha-Hb and fli1a (48 hpf) as well as rag2 and lck (4 days post-fertilization), and the expression of spi1b and mpo decreased (48 hpf). The expression of primitive hematopoietic stem cell markers scl and lmo2, as well as dnmt3ab, beta-Hb, l-plastin, and rag1 were unaffected. The levels of 5-mC and 5-hmC measured by ELISA were also decreased after MO knock-down of tet2. The number of gata-1 expressing red blood cells increased after tet2 MO knock-down as evaluated by flow-cytometry indicating that tet2 deficiency increased erythropoiesis. These preliminary results suggest that tet2 might play a role in the epigenetic regulation of zebrafish early hematopoiesis including erythropoiesis. Recently, transcription activator-like effector nuclease (TALEN) has been shown to generate targeted genomic editing in zebrafish. To validate our observation, we therefore utilized customized TALENs pair to generate tet2 knock-out zebrafish animal model. We designed a pair of TALENs targeting first exon of tet2 and our tet2 TALENs were able to generate insertion and/or deletion in targeted region of tet2 exon 1 in 25% to 44% zebrafish embryos. We obtained a total of fifteen different tet2 mutation genotypes F1 fish, and seven of them were predicted to cause early termination of transcription. The in-cross of these F1 genotypes matched the Mendelian inheritance. The tet2-/- knock-out F2 zebrafish is not embryonic lethal and can grow to sexually mature adult fish. The detailed analysis of tet2-/- knock-out zebrafish early hematopoiesis will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

Lymphoid Precursors Arise Independently at Multiple Sites During Development.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 699-699
Author(s):  
Prashanth Porayette ◽  
Momoko Yoshimoto ◽  
Nicole Glosson ◽  
Michael Ferkowicz ◽  
William C. Shelley ◽  
...  
Keyword(s):  
T Cells ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Γδ T Cells ◽  
Systemic Circulation ◽  
Early Embryo ◽  
Lymphoid Cells ◽  
Recipient Mouse ◽  
Hematopoietic Stem ◽  
Knock Out

Abstract Abstract 699 Primitive hematopoietic cells arise on embryonic day 7.0 (E7.0) within the murine extra-embryonic yolk sac (YS) followed by formation of definitive hematopoietic cells on E8.25. The first cells capable of long term multi-lineage repopulation in an adult irradiated mouse arise in the embryo proper on E10.5. The temporal and spatial emergence of lymphoid precursors capable of forming mature T and B lymphocytes remains controversial. Cardiac contractions begin on E8.25, leading to the circulation of cells between various sites of hematopoiesis thus making it difficult to identify definitively the site where lymphoid precursors emerge. We have previously utilized a transgenic knock out murine mutant in which no heartbeat develops to examine hematopoietic cell emergence. NCX1 is a protein required for the initiation of cardiac contractions. The ncx1 null embryos develop normally until about E10.5, and then succumb to the lack of a systemic circulation. Single cell suspensions from within the para-aortic splanchnopleure (P-Sp) of the embryo proper and from YS at E8.5 and E9.5 were isolated and plated on the OP9-Dl1 stromal cell line that is known to support T lymphopoiesis. We observed that both YS and P-Sp independently displayed the potential to produce mature T cells including CD4 and CD8 DN, DP, and SP cells. We observed that both sites can give rise to both αaβ and γδ T cells independently at a frequency approaching 1:165 hemogenic endothelial cells plated. Cells derived from both YS and P-Sp demonstrated lymphoid specific VDJ rearrangement. Adoptive transfer of the lymphoid cells derived from these sites into NOD/SCID/IL2Rg null recipient mice revealed engraftment within multiple lymphohematopoietic organs including thymus, spleen and liver in the recipient mouse and the presence of circulating donor T cells. Stimulation of splenocytes from transplanted animals with anti-CD3 antibodies induced proliferation of these cells suggesting intact antibody responses in YS and P-Sp derived T cells. Development of mature functional T lymphocytes from precursors isolated from YS and P-Sp before the presence of a functional thymus brings forward the hypothesis for an extra-thymic multi-site origin of T cells in the early embryo. The presence of prospective lymphoid precursors more than a day earlier than the adult reconstituting hematopoietic stem cell (HSC) first observed at E10.5 during development suggests that either the HSC may be present at earlier points in development than is believed or that there might be a HSC independent emergence of lymphoid precursors in the developing embryo. Disclosures: No relevant conflicts of interest to declare.

S1P1 Agonists for Use as Adjunct Mobilizing Agents

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 826-826 ◽  
Author(s):  
Nadia Harun ◽  
Marilyn Thien ◽  
Julius G Juarez ◽  
Kenneth Francis Bradstock ◽  
Linda J. Bendall
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Receptor Expression ◽  
Hematopoietic Stem ◽  
Knock Out ◽  
S1p Receptor ◽  
Knock Out Mouse

Abstract Abstract 826 Harvesting hematopoietic stem cells (HSC) mobilised into peripheral blood (PB) for transplantation is mediated through bone marrow (BM) retentive and egress factors. Factors that retain hematopoietic stem cells (HSC) in the BM are well defined, with CXCL12 and VCAM1 playing major roles. However, the factors involved in the egress of HSC from the BM into the peripheral blood (PB) are currently uncharacterised. Sphingosine-1-Phosphate (S1P) is a lymphoid organ egress factor for lymphocytes, mediated through the S1P1 receptor, which is also expressed on HSC. We hypothesised that S1P mediates the egress of HSC out of the BM and into the PB. Our laboratory used a number of different mouse models with various S1P levels or S1P receptor expression to elucidate the role of the S1P gradient between the BM and PB. Sphingosine kinase-1 knock-out (SK1KO) mice were utilized for their reduced PB S1P levels. A sphingosine lyase inhibitor 4′deoxypyridoxine (DOP) was used to increase BM S1P levels. Mice treated with FTY720 for 14h had suppressed S1P1 expression and an S1P1 conditional knock-out mouse was also generated by our group. Animals were also treated with S1P receptor agonists such as SEW2871. Mobilisation experiments, competitive repopulation assays and chemotaxis assays were performed utilizing the various models. Plasma from SK1KO mice had a reduced capacity to induce migration in haematopoietic progenitor cells (HPC), confirming the chemokine activity of S1P. Consistent with this, AMD3100 induced mobilization was inhibited in SK1KO mice and DOP treated mice, demonstrating the role of an S1P gradient in HPC mobilization. Mice treated with FTY720 significantly inhibited AMD3100, although not G-CSF, mediated mobilisation of HPC in mice. No HPC accumulation was detected in secondary lymphoid organs such as lymph nodes or spleen. Most importantly, FTY720 treatment reduced the number of transplantable HSC in the blood following AMD3100-mediated mobilisation using a competitive repopulation assay. Our laboratory also generated an S1P1 conditional knock-out mouse. When mobilised with AMD3100, these S1P1 knock-out animals displayed a marked reduction in HPC mobilisation compared to wild-type animals. Finally, the S1P1 agonist SEW2871 increased HPC mobilisation synergistically, by approximately 2 fold when combined with AMD3100, but not G-CSF. S1P supports the egress of HSC from the BM into the PB following inhibition of the CXCL12/CXCR4 axis. S1P1 conditional knock-out mice display a significantly reduced mobilising capacity. S1P receptor agonist, SEW2871, acts synergistically with AMD3100 to increase HPC mobilisation in vivo, raising the possibility that such a combination may increase the efficiency of HSC collection for transplantation purposes. Disclosures: No relevant conflicts of interest to declare.

Microrna-155 Promotes Hematopoietic Stem and Progenitor Cell Mobilization and Proliferation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 214-214
Author(s):  
Tomer Itkin ◽  
Aya Ludin ◽  
Shiri Gur-Cohen ◽  
Carolin Ludwig ◽  
Robert Brooks ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Expression Patterns ◽  
Hematopoietic Cells ◽  
Wild Type ◽  
Cell Frequency ◽  
Hematopoietic Stem ◽  
Knock Out ◽  
Cell Counts ◽  
Intracellular Response

Abstract Abstract 214 MicroRNAs (miRNAs) are small non-coding RNAs involved in various physiological processes, including hematopoiesis. Although miRNAs are broadly studied with regards to normal and malignant leukocyte development, the role of miRNAs in hematopoietic stem and progenitor (HSPC) migration and mobilization is poorly understood. Currently, induction of HSPC mobilization from the bone marrow (BM) to the peripheral blood (PB) is the major mean to harvest HSPCs for clinical transplantation. Recently, several miRNAs were found to be upregulated in macaque G-CSF-mobilized CD34+ HSPCs, among them the oncogenic miRNA mir-155 (Donahue et al., Blood 2009). To study the involvement of mir-155 in HSPC regulation, we examined hematopoiesis in mir-155 knock out (KO) mice. Of interest, mir-155 KO mice had normal BM and PB levels of mature cells, but reduced levels of immature BM Lineage−/Sca-1+/c-Kit+ (LSK) and primitive BM CD34−LSK HSPCs. Profiling of mir-155 expression in murine hematopoietic BM populations, following G-CSF treatment, revealed differential expression patterns in wild type (WT) mice. G-CSF treatment upregulated mir-155 levels in immature LSK cells, in T-cells and in Mac-1+/Gr-1+ monocyte/macrophages. In contrast, G-CSF downregulated mir-155 levels in common lymphoid progenitors and in B-cells. Suggesting that mature hematopoietic cells may also participate in HSPC mobilization process. G-CSF administration to mir-155 KO mice resulted in reduced HSPC mobilization, as assessed by CFU-C and LSK cell counts in the PB. Surprisingly, G-CSF treatment increased BM LSK cell frequency in mir-155 KO mice to the same levels as in WT mice. On the contrary, G-CSF treatment reduced BM CD34−LSK cell frequency in WT mice and increased it in mir-155 KO mice showing an opposing effect on the more primitive HSPC population. Since mir-155 is involved also in mesenchymal development regulating osteoblast differentiation, we propose that BM HSPC pool reduction could be mediated by the stromal microenvironment. Additionally, osteoblasts and other BM residing cells undergo substantial changes in response to G-CSF that might be mediated by mir-155. To determine whether the mobilization defect is hematopoietic cell-autonomous or due to an abnormal microenvironment, we examined G-CSF-induced mobilization in chimeric mice. Mir-155 KO mice reconstituted with wild type (WT) BM cells had normal mobilization as WT mice reconstituted with WT BM cells. Of interest, WT mice reconstituted with mir-155 KO BM cells showed reduced mobilization as mir-155 KO mice reconstituted with mir-155 KO BM cells. These results indicate that the mobilization defect in mir-155 KO mice is also due to a defect in HSPC motility. Since the CXCL12/CXCR4 axis plays a major role in HSPC mobilization, we examined the ability of mir-155 KO cells to perform CXCL12-induced migration and found reduced migration capacity of HSPCs in vitro. Although having reduced migration potential, mir-155 KO LSK cells had normal CXCR4 expression levels, suggesting that an aberrant intracellular response to SDF-1 is responsible for the observed defect. In support, AMD3100 treatment to mir-155 KO mice resulted in reduced HSPC rapid mobilization. Since SHIP-1 phosphatase mRNA is targeted by mir-155 in hematopoietic cells (Costinean et al., Blood 2009) and SHIP-1 KO hematopoietic cells exhibit increased migration towards CXCL12 (Kim et al., JCI 1999), we examined intracellular SHIP-1 expression during HSPC mobilization. SHIP-1 levels were downregulated in WT BM LSK cells in response to G-CSF or AMD3100 mobilizing treatments. In contrast, mir-155 KO BM LSK cells upregulated SHIP-1 levels in response to the mobilizing treatments. These results suggest that mir-155 may promote HSPC mobilization and increased motility via SHIP-1 downregulation. In summary, our data indicates that mir-155 directly promotes HSPC motility and mobilization by SHIP-1-mediated regulation of intracellular response to CXCL12 signaling. We also propose the mechanism of indirect regulation of BM HSPC pool size during steady state and following G-CSF treatment by mir-155, via stromal BM microenvironment, which is currently under investigation. Deciphering the mechanisms of HSPC migration and maintenance in general and by mir-155 in particular, may potentially improve clinical mobilization protocols and contribute to increased donor BM engraftment following transplantation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Autophagy and Metabolism in Normal and Malignant Hematopoiesis

2021 ◽  
Vol 22 (16) ◽  
pp. 8540
Author(s):  
Ioanna E. Stergiou ◽  
Efstathia K. Kapsogeorgou
Keyword(s):  
Hematopoietic Cells ◽  
Building Blocks ◽  
Hematologic Malignancies ◽  
Mitochondrial Content ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Regulation Of Metabolism ◽  
Multipotent Differentiation

The hematopoietic system relies on regulation of both metabolism and autophagy to maintain its homeostasis, ensuring the self-renewal and multipotent differentiation potential of hematopoietic stem cells (HSCs). HSCs display a distinct metabolic profile from that of their differentiated progeny, while metabolic rewiring from glycolysis to oxidative phosphorylation (OXPHOS) has been shown to be crucial for effective hematopoietic differentiation. Autophagy-mediated regulation of metabolism modulates the distinct characteristics of quiescent and differentiating hematopoietic cells. In particular, mitophagy determines the cellular mitochondrial content, thus modifying the level of OXPHOS at the different differentiation stages of hematopoietic cells, while, at the same time, it ensures the building blocks and energy for differentiation. Aberrations in both the metabolic status and regulation of the autophagic machinery are implicated in the development of hematologic malignancies, especially in leukemogenesis. In this review, we aim to investigate the role of metabolism and autophagy, as well as their interconnections, in normal and malignant hematopoiesis.

Constitutive TGF-β1 Deficiency Induces a Defect in Hematopoietic Stem/Progenitor Cell Compartment in Fetus and Neonate.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1397-1397
Author(s):  
Claude Capron ◽  
Catherine Lacout ◽  
Yann Lecluse ◽  
Valérie Jalbert ◽  
Elisabeth Cramer Bordé ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Type I ◽  
Activated T Cells ◽  
Hematopoietic Stem ◽  
Tgf Β1

Abstract TGF-β1 is a cytokine with pleiotropic effects. It has been considered that TGF-β1plays a major role on hematopoietic stem cells (HSC) based on in vitro experiment. Achieving in vivo experiments proved to be difficult because constitutive TGF-β1 knock-out (KO) in mice leads to lethality during the first 4 weeks of life from a wasting syndrome related to tissue infiltration by activated T cells and macrophages. For this reason, hematopoiesis of TGF-β1−/− mice has not been studied in details. In contrast the role of TGF-β1 has been recently extensively studied in conditional TGF-β type I receptor (TβRI) KO mice. No clear effect was observed on HSC functions, suggesting that TGF-β1 was not a key physiological regulator of hematopoiesis in the adult. However, these experiments have some limitations. They do not exclude a putative role for TGF-β1 during fetal hematopoiesis and they do not specifically address the role of TGF-β1 on hematopoiesis because KO of TGF-β receptor leads to signaling arrest for all TGF-βs. In addition, other receptors may be involved in TGF-β1 signaling. For these reasons, we have investigated the hematopoiesis of constitutive TGF-β1 KO mice with a mixed Sv129 × CF-1 genetic background allowing the birth of a high proportion of homozygotes. In 2 week-old neonate mice, we have shown a decrease of bone marrow (BM) and spleen progenitors and a decrease of immature progenitors colony forming unit of the spleen (CFU-s). Moreover this was associated with a loss in reconstitutive activity of TGF-β1−/− HSC from BM. However, although asymptomatic, these mice had an excess of activated lymphocytes and an augmentation of Sca-1 antigen on hematopoietic cells suggesting an excess of γ-interferon release. Thus we studied hematopoiesis of 7 to 10 days-old neonate mice, before phenotypic modification and inflammatory cytokine release. Similar results were observed with a decrease in the number of progenitors and in the proliferation of TGF-β1−/− BM cells along with an increased differentiation but without an augmentation in apoptosis. Moreoever, a loss of long term reconstitutive capacity of BM Lineage negative (Lin−) TGF-β1−/− cells along with a diminution of homing of TGF-β1−/− progenitors was found. These results demonstrate that TGF-β1 may play a major role on the HSC/Progenitor compartment in vivo and that this defect does not seem to be linked to the immune disease. To completely overpass the risk of the inflammatory syndrome, we analyzed hematopoiesis of fetal liver (FL) of TGF-β1−/− mice and still found a decrease in progenitors, a profound defect in the proliferative capacities, in long term reconstitutive activity and homing potential of primitive FL hematopoietic cells. Our results demonstrate that TGF-β1 plays an important role during hematopoietic embryonic development. Altogether these findings suggest that TGF-β1 is a potent positive regulator for the in vivo homeostasis of the HSC compartment.

Aberrant Bone Marrow Perivascular Niches are Involved in Multiple Myeloma Progression: Role of Notch–Delta Pathway.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2800-2800
Author(s):  
Sara Lamorte ◽  
Marta Costa ◽  
Giovanni Camussi ◽  
Sergio Dias
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Pcr Analysis ◽  
Adherent Cells ◽  
Feeder Layer ◽  
Hematopoietic Stem ◽  
Lower Chamber

Abstract Abstract 2800 Poster Board II-776 Bone marrow (BM) angiogenesis is implicated in Multiple Myeloma (MM) progression. In this study, we tested the hypothesis that MM progression occurs when aberrant BM perivascular niches are established. We isolated BM endothelial cells derived from MM patients (MM-BMECs) from BM aspirates using anti-CD31Ab coupled to magnetic beads. FACS analysis showed that of all the cell lines isolated were endothelial: more than 95% expressed Ulex Europaeus Agglutinin-1 and Factor VIII and were negative for monocyte-macrophage (CD14) and plasma cell markers (CD38). To test the hypothesis that in MM patients BM perivascular niches are aberrant we analyzed how MM-BMECs modulate hematopoietic stem cells (HSCs) properties using a BM microvascular endothelial cell line isolated from a healthy donor (BMECs) as control. We co-cultured cord blood cells CD34+ HSCs in the presence of MM-BMECs or BMECs feeder layer and we analyzed the ability of MM-BMECs compared with BMECs to modulate HSCs adhesion, chemotaxis and apoptosis. The results show that MM-BMECs promote CD34+ HSCs adhesion, recruitment and protect them from apoptosis. In detail, we showed that after 24h of co-culture there was a significant increase in the number of adherent HSCs on MM-BMECs than on BMECs: 43±9% versus 25±6%. Moreover, when HSCs were cultured for 48 hours in 1% of serum in the presence of MM-BMECs they were less sensitive to apoptosis (9±11% of Annexin V+ cells) than HSCs cultured in the presence of BMECs (14±1% of Annexin V+ cells) or without a feeder layer, as control (17±3% of Annexin V+ cells). For the migration assay a transwell chamber system, in which the upper and the lower chambers were separated by 5-μm pore-size filter, was used. BMECs, MM-BMECs or nothing was plated in the lower chamber, while HSCs were seeded into the upper chamber. Both chambers were loaded with unsupplemented EBM-2 plus 2% of serum. Cell migration was studied over a 6-8 hours period and evaluated as number of cells migrated into the lower chamber. The results showed a significantly greater migration of HSCs in the presence of MM-BMECs than BMECs: 12±2% versus 5±1% of migrated cells. Taken together, these data showed that MM-BMECs promoted HSCs migration, adhesion and survival. Next we evaluated how MM-BMECs modulate the hemopoiesis recovery after irradiation in a NOD-SCID mouse model. When injected into sub-lethally irradiated (3 Grey) NOD-SCID mice MM-BMECs were detected in the BM integrated within the murine BM vessels and promoted hematopoietic recovery. In detail, MM-BMECs provided signals favoring the commitment towards lymphoid lineage. In fact, 7 days after injection, the BM of mice injected with MM-BMECs showed an increase in the percentage of lymphoblast (2.7%), compared with mice injected with BMECs or PBS, as control (respectively, 1.5% and 1.4%); followed, 14 days after injection, by a significant increase in the percentage of peripheral blood lymphocytes in mice injected with MM-BMECs (75±6%) versus mice injected with BMECS and PBS (respectively 60±0.5% and 47±7%). Since MM is a plasma cells disorder and the Notch-Delta pathway has been shown to play a central role in regulating HSCs properties, including the decisions of HSCs to undergo T- or B-cell differentiation, we investigated the involvement of this pathway in MM-BMECs and HSCs interaction. As determined by FACS and RT-PCR analysis, MM-BMECs, compared to BMECs, over expressed Delta-like Notch ligand 4 (DII4). Thus, we investigated the role of DII4 in the MM-BMECs/BMECs-HSCs adhesion. The first results showed that the expression of DII4 by MM-BMECs is necessary to promote HSCs adhesion. In fact, using a blocking antibody against DII4 (AbαDII4) at 50ug/ml there was an impairment in HSCs adhesion to MM-BMECs (43±9% versus 24±2% of adherent cells without and with AbαDII4 treatment), but not on BMECs (25±6% versus 26±1.4% of adherent cells without and with AbαDII4 treatment). Ongoing experiments are focusing on the role of DII4 in the modulation of HSCs proliferation, protection against apoptosis and in vitro-in vivo B commitment by MM-BMECs. Taken together, all these data suggest that BMECs in MM may function as “aberrant perivascular niches”, modulating HSCs properties. This aberrant phenotype could be due to an alteration of the Notch-Delta pathway in BMECs that favors malignant clonal growth by protecting it from apoptosis, favoring migration, adhesion and providing self-renewing and/or proliferative cues. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 367-367
Author(s):  
Marcin Wysoczynski ◽  
HakMo Lee ◽  
Rui Liu ◽  
Wan Wu ◽  
Janina Ratajczak, ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Membrane Attack Complex ◽  
Classical Pathway ◽  
Compensatory Mechanism ◽  
Complement Cascade ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Deficient Mice

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

Poly I:C Stimulates Sca-1 Expression in Murine Bone Marrow and Increases the Number of Phenotypic Hematopoietic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2504-2504
Author(s):  
Russell Garrett ◽  
Gerd Bungartz ◽  
Alevtina Domashenko ◽  
Stephen G. Emerson
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Poly I:C ◽  
Hematopoietic Stem ◽  
Marrow Cells ◽  
Myeloid Progenitors

Abstract Abstract 2504 Poster Board II-481 Polyinosinic:polycytidlyic acid (poly I:C) is a synthetic double-stranded RNA used to mimic viral infections in order to study immune responses and to activate gene deletion in lox-p systems employing a Cre gene responsive to an Mx-1 promoter. Recent observations made by us and others have suggested hematopoietic stem cells, responding to either poly I:C administration or interferon directly, enter cell cycle. Twenty-two hours following a single 100mg intraperitoneal injection of poly I:C into 10-12 week old male C57Bl/6 mice, the mice were injected with a single pulse of BrdU. Two hours later, bone marrow was harvested from legs and stained for Lineage, Sca-1, ckit, CD48, IL7R, and BrdU. In two independent experiments, each with n = 4, 41 and 33% of Lin- Sca-1+ cKit+ (LSK) IL-7R- CD48- cells from poly I:C-treated mice had incorporated BrdU, compared to 7 and 10% in cells from PBS-treated mice. These data support recently published reports. Total bone marrow cellularity was reduced to 45 and 57% in the two experiments, indicating either a rapid death and/or mobilization of marrow cells. Despite this dramatic loss of hematopoietic cells from the bone marrow of poly I:C treated mice, the number of IL-7R- CD48- LSK cells increased 145 and 308% in the two independent experiments. Importantly, the level of Sca-1 expression increased dramatically in the bone marrow of poly I:C-treated mice. Both the percent of Sca-1+ cells and the expression level of Sca-1 on a per cell basis increased after twenty-four hours of poly I:C, with some cells acquiring levels of Sca-1 that are missing from control bone marrow. These data were duplicated in vitro. When total marrow cells were cultured overnight in media containing either PBS or 25mg/mL poly I:C, percent of Sca-1+ cells increased from 23.6 to 43.7%. Within the Sca-1+ fraction of poly I:C-treated cultures, 16.7% had acquired very high levels of Sca-1, compared to only 1.75% in control cultures. Quantitative RT-PCR was employed to measure a greater than 2-fold increase in the amount of Sca-1 mRNA in poly I:C-treated cultures. Whereas the numbers of LSK cells increased in vivo, CD150+/− CD48- IL-7R- Lin- Sca-1- cKit+ myeloid progenitors almost completely disappeared following poly I:C treatment, dropping to 18.59% of control marrow, a reduction that is disproportionately large compared to the overall loss of hematopoietic cells in the marrow. These cells are normally proliferative, with 77.1 and 70.53% accumulating BrdU during the 2-hour pulse in PBS and poly I:C-treated mice, respectively. Interestingly, when Sca-1 is excluded from the analysis, the percent of Lin- IL7R- CD48- cKit+ cells incorporating BrdU decreases following poly I:C treatment, in keeping with interferon's published role as a cell cycle repressor. One possible interpretation of these data is that the increased proliferation of LSK cells noted by us and others is actually the result of Sca-1 acquisition by normally proliferating Sca-1- myeloid progenitors. This new hypothesis is currently being investigated. Disclosures: No relevant conflicts of interest to declare.

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