Balanced Wnt5a-Mediated Signaling Is Necessary for Normal Proliferation of Primitive Hematopoietic Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2533-2533
Author(s):  
Benjamin Povinelli ◽  
Michael Baranello ◽  
Kathleen Kokolus ◽  
Michael Nemeth
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transcription Factors ◽  
Developmental Stage ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Cells Cultured ◽  
Marrow Cells

Abstract Abstract 2533 Poster Board II-510 Multiple members of the Wnt family of ligands have been implicated in the regulation of self-renewal and proliferation of hematopoietic stem cells (HSCs). Previously, we have observed that ex vivo expansion of HSCs in the presence of recombinant murine Wnt5a (rmWnt5a) resulted in increased hematopoietic repopulation. Based on these data, we hypothesized that Wnt5a is necessary for normal function of HSCs and hematopoietic progenitors (HPCs). Since Wnt5a deficiency (Wnt5a−/−) is perinatal lethal in vivo, we tested this hypothesis using in vitro Dexter stroma cultures established using whole bone marrow. To determine the ability of Wnt5a to support hematopoiesis in the context of the adult hematopoietic microenvironment, we cultured lineage-negative (lin−) HPCs on irradiated bone marrow stroma in the presence of 5 μg/ml Wnt5a-neutralizing antibody (Wnt5a-Ab). After two weeks, we observed that hematopoietic cells cultured on untreated stroma contained 4.8-fold more myeloid CFU (33.1 ± 12.3 CFU/104 cells) than cells cultured on Wnt5a-Ab stroma (6.9 ± 0.7 CFU; n = 3, p < .01). A similar difference was observed after 4 weeks (control: 16.0 ± 7.2 CFU/104 cells; Wnt5a-Ab: 1.3 ± 2.3 CFU; n = 3; p = .03). In the converse experiment, lin− HPCs were cultured on stroma in the presence of Wnt5a conditioned medium (Wnt5a-CM). We observed after two weeks that hematopoietic cells cultured on stroma with control-CM contained 4.4-fold more myeloid CFU (29.8 ± 13.5 CFU/104 cells) than cells cultured on Wnt5a-CM stroma (6.8 ± 2.2 CFU; n = 4, p = .02). Together, these data indicate that Wnt5a-mediated signaling must be balanced in order for normal hematopoiesis to occur. To determine if the effects of Wnt5a required the presence of developmental stage-specific factors, we established stroma cultures from fetal spleens harvested from E17.5 Wnt5a−/− mice and littermate controls and seeded them with lin− HPCs. We observed that hematopoietic cells cultured on control fetal spleen stroma contained 12.5-fold more myeloid CFU (72.6 ± 21.6 CFU/104 cells) than cells cultured on Wnt5a−/− fetal spleen stroma (5.8 ± 5.8; n = 6, p < .001) after two weeks. These data indicate that the effect of Wnt5a on hematopoietic cells is independent of the developmental stage of the surrounding microenvironment. To determine if the effects of Wnt5a were due to regulation of proliferation or differentiation of hematopoietic cells, we cultured bone marrow cells in cytokine-supplemented methylcellulose in the presence of 300 ng/ml rmWnt5a. We observed a 5.5-fold decrease in the number of myeloid CFU formed in cultures with rmWnt5a (14.1 ± 3.8/104 cells) compared to control (77.6 ± 5.1 CFU; n = 3; p < .001), suggesting that Wnt5a could be regulating both processes. In the converse experiment, we cultured bone marrow cells for 4 days in cytokine-supplemented serum-free media with the same dose of rmWnt5a after which equal numbers of cells were plated in rmWnt5a-free methylcellulose. We did not observe any difference in CFU frequency between control (19.3 ± 4.2 CFU/104 cells) and rmWnt5a (24.0 ± 2.6 CFU; n = 3) cultures, indicating that treatment with rmWnt5a inhibited hematopoietic proliferation but not differentiation. To identify the mechanism by which Wnt5a regulates HSC and HPC proliferation, we analyzed potential Wnt5a-mediated signaling pathways. We observed that Wnt5a induced intracellular Ca2+ (iCa2+) flux in HSCs (defined as lin−, Sca-1HI, c-kitHI; LSK). Previous studies have shown that Wnt5a-mediated induction of iCa2+ can result in activation of the NFAT family of transcription factors. Since NFATc1 promotes quiescence of hair follicle stem cells and is expressed in HSCs, we hypothesized that the effects of Wnt5a required activation of NFAT family members. We cultured bone marrow cells with Wnt5a-CM in the presence of cyclosporine A (CsA), which inhibits activation of NFAT factors. In agreement with our earlier findings, we observed that culturing bone marrow cells in Wnt5a-CM increased the percentage of quiescent (defined as Ki-67-) LSKCD34− HSCs (79.7 ± 3.3%) compared to control-CM (55.0 ± 1.6%; n = 3; p < .001). This increase was inhibited by CsA (69.4 ± 2.6%; n = 3; p = .01 compared to Wnt5a-CM alone). In conclusion, our data point to a role for Wnt5a in regulating HSC and HPC proliferation and that this function may require the activation of NFAT transcription factors. 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.

Implication of AML1/RUNX1 Function in the Homeostasis and Leukemic Transformation of Hematopoietic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4117-4117
Author(s):  
Motoshi Ichikawa ◽  
Masataka Takeshita ◽  
Susumu Goyama ◽  
Takashi Asai ◽  
Eriko Nitta ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Chimeric Protein ◽  
Chromosomal Translocation ◽  
Hematopoietic Stem ◽  
Quiescent Hscs ◽  
Marrow Cells

Abstract Transcription factor AML1/RUNX1, initially isolated from the t(8;21) chromosomal translocation in human leukemia, is essential for the development of multilineage hematopoiesis in mouse embryos. AML1 negatively regulates the number of immature hematopoietic cells in adult hematopoiesis, while it is required for megakaryocytic maturation and lymphocytic development. However, it remains yet to be determined how AML1 contributes to homeostasis of hematopoietic stem cells (HSCs). To address this issue, we analyzed in detail HSC function in the absence of AML1. Notably, cells in the Hoechst 33342 side population fraction and c-Kit-positive cells in the G0 cell cycle status were increased in number in AML1-deficient bone marrow, which suggests enrichment of quiescent HSCs. We also found an increase in HSC number within the AML1-deficient bone marrow using limiting dilution bone marrow transplantation assays. Thus, the number of quiescent HSCs is negatively regulated by AML1, loss of which may result in accumulation of leukemic stem cell pool in AML1-related leukemia. To identify mechanisms through which functional loss of AML1 exerts leukemogenic potential, we focused on the AML1-Evi-1 chimeric protein, which is generated by the t(3;21) chromosomal translocation and disturbs the normal function of AML1. We introduced AML1-Evi-1 and its mutants into murine bone marrow cells, and evaluated hematopoietic cell transformation by colony replating assays. The transforming activity of AML1-Evi-1 was impaired when any of the major functional domains of AML1-Evi-1 was lost. Moreover, overexpression of Evi-1 could not transform AML1-deleted bone marrow cells, suggesting that fusion of AML1 and Evi-1, rather than AML1 suppression and Evi-1 overexpression, is essential for AML1-Evi-1 leukemogenesis. Intriguingly, among the hematopoietic progenitor cell fractions, AML1-Evi-1 could transform only the uncommitted, immature hematopoietic cells, which contrasts with MLL-ENL, a chimeric protein generated in t(11;19) leukemia. AML1-Evi-1 transformed cells show a surface marker profile different from that of the cells transformed by AML1-MTG8/ETO, another leukemic gene product that also perturbs AML1 function. These results provide a valuable clue to a distinct mechanism determined by the Evi-1 moiety in the AML1-Evi-1 leukemogenesis and to a role of AML1 loss in the self-renewal of leukemic stem cells.

scholarly journals Development of a Novel Selective Amplifier Gene for Controllable Expansion of Transduced Hematopoietic Cells

Blood ◽  
1997 ◽  
Vol 90 (10) ◽  
pp. 3884-3892 ◽  
Author(s):  
Keiko Ito ◽  
Yasuji Ueda ◽  
Masaki Kokubun ◽  
Masashi Urabe ◽  
Toshiya Inaba ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Selective Amplifier ◽  
Marrow Cells

Abstract To overcome the low efficiency of gene transfer into hematopoietic cells, we developed a novel system for selective expansion of transduced cells. To this end, we constructed a chimeric cDNA (GCRER) encoding the fusion protein between the granulocyte colony-stimulating factor receptor (G-CSFR) and the hormone-binding domain (HBD) of the estrogen receptor (ER) as a selective amplifier gene. Use of the intracellular signaling pathway of G-CSFR was considered to be appropriate, because G-CSF has the ability not only to stimulate the neutrophil production, but also to expand the hematopoietic stem/progenitor cell pool in vivo. To activate the exogenous G-CSFR signal domain selectively, the estrogen/ER-HBD system was used as a molecular switch in this study. When the GCRER gene was expressed in the interleukin-3 (IL-3)–dependent murine cell line, Ba/F3, the cells showed IL-3–independent growth in response to G-CSF or estrogen. Moreover, the Ba/F3 cells transfected with the Δ(5-195)GCRER, whose product lacks the extracellular G-CSF–binding domain, did not respond to G-CSF, but retained the ability for estrogen-dependent growth. Further, murine bone marrow cells transduced with the GCRER or Δ(5-195)GCRER gene with retroviral vectors formed a significant number of colonies in response to estrogen, as well as G-CSF, whereas estrogen did not stimulate colony formation by untransduced murine bone marrow cells. It is noteworthy that erythroid colonies were apparently formed by the bone marrow cells transduced with the GCRER gene in the presence of estrogen without the addition of erythropoietin, suggesting that the signals from the G-CSFR portion of the chimeric molecules do not preferentially induce neutrophilic differentiation, but just promote the differentiation depending on the nature of the target cells. We speculate that when the selective amplifier genes are expressed in the primitive hematopoietic stem cells, the growth signal predominates and that the population of transduced stem cells expands upon estrogen treatment, even if some of the cells enter the differentiation pathway. The present study suggests that this strategy is applicable to the in vivo selective expansion of transduced hematopoietic stem cells.

scholarly journals MicroRNA-126 Regulates HOXA9 by Binding to the Homeobox

2008 ◽  
Vol 28 (14) ◽  
pp. 4609-4619 ◽  
Author(s):  
Wei-Fang Shen ◽  
Yu-Long Hu ◽  
Lalita Uttarwar ◽  
Emmanuelle Passegue ◽  
Corey Largman
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Biological Activity ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
F9 Cells ◽  
Functional Conservation ◽  
Marrow Cells

ABSTRACT The PicTar program predicted that microRNA-126 (miR-126), miR-145, and let-7s target highly conserved sites within the Hoxa9 homeobox. There are increased nucleotide constraints in the three microRNA seed sites among Hoxa9 genes beyond that required to maintain protein identity, suggesting additional functional conservation. In preliminary experiments, forced expression of these microRNAs in Hoxa9-immortalized bone marrow cells downregulated the HOXA9 protein and caused loss of biological activity. The microRNAs were shown to target their predicted sites within the homeobox. miR-126 and Hoxa9 mRNA are coexpressed in hematopoietic stem cells and downregulated in parallel during progenitor cell differentiation; however, miR-145 is barely detectable in hematopoietic cells, and let-7s are highly expressed in bone marrow progenitors, suggesting that miR-126 may function in normal hematopoietic cells to modulate HOXA9 protein. In support of this hypothesis, expression of miR-126 alone in MLL-ENL-immortalized bone marrow cells decreased endogenous HOXA9 protein, while inhibition of endogenous miR-126 increased expression of HOXA9 in F9 cells.

scholarly journals Long-term monoclonal reconstitution of erythropoiesis in genetically anemic W/Wv mice by injection of 5-fluorouracil-treated bone marrow cells of Pgk-1b/Pgk-1a mice

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1758-1763 ◽  
Author(s):  
T Nakano ◽  
N Waki ◽  
H Asai ◽  
Y Kitamura
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Electrophoretic Pattern ◽  
Phosphoglycerate Kinase ◽  
X Chromosomes ◽  
Hematopoietic Stem ◽  
Colony Forming Units ◽  
Marrow Cells

Abstract The spleen colony-forming assay does not represent the number of hematopoietic stem cells with extensive self-maintaining capacity because five to 50 spleen colony-forming units (CFU-S) are necessary to rescue a genetically anemic (WB X C57BL/6)F1-W/Wv(WBB6F1-W/Wv) mouse. We investigated which is more important for the reconstitution of erythropoiesis, the transplantation of multiple CFU-S or that of a single stem cell with extensive self-maintaining potential. The electrophoretic pattern of hemoglobin was used as a marker of reconstitution and that of phosphoglycerate kinase (PGK), an X chromosome-linked enzyme, as a tool for estimating the number of stem cells. For this purpose, we developed the C57BL/6 congeneic strain with the Pgk-1a gene. Bone marrow cells were harvested after injection of 5- fluorouracil from C57BL/6-Pgk-1b/Pgk-1a female mice in which each stem cell had either A-type PGK or B-type PGK due to the random inactivation of one or two X chromosomes. When a relatively small number of bone marrow cells (ie, 10(3) or 3 X 10(3] were injected into 200-rad- irradiated WBB6F1-W/Wv mice, the hemoglobin pattern changed from the recipient type (Hbbd/Hbbs) to the donor type (Hbbs/Hbbs) in seven of 150 mice for at least 8 weeks. Erythrocytes of all these WBB6F1-W/Wv mice showed either A-type PGK alone or B-type PGK alone during the time of reconstitution, which suggests that a single stem cell with extensive self-maintaining potential may sustain the whole erythropoiesis of a mouse for at least 8 weeks.

Enforced Expression of the GATA-2 Transcription Factor Blocks Normal Hematopoiesis

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 488-499 ◽  
Author(s):  
Derek A. Persons ◽  
James A. Allay ◽  
Esther R. Allay ◽  
Richard A. Ashmun ◽  
Donald Orlic ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Blood Cell ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Immunoblot Analysis ◽  
Marrow Cells

Abstract The zinc finger transcription factor GATA-2 is highly expressed in immature hematopoietic cells and declines with blood cell maturation. To investigate its role in normal adult hematopoiesis, a bicistronic retroviral vector encoding GATA-2 and the green fluorescent protein (GFP) was used to maintain the high levels of GATA-2 that are normally present in primitive hematopoietic cells. Coexpression of the GFP marker facilitated identification and quantitation of vector-expressing cells. Bone marrow cells transduced with the GATA-2 vector expressed GFP as judged by flow cytometry and GATA-2 as assessed by immunoblot analysis. A 50% to 80% reduction in hematopoietic progenitor-derived colony formation was observed with GATA-2/GFP-transduced marrow, compared with marrow transduced with a GFP-containing vector lacking the GATA-2 cDNA. Culture of purified populations of GATA-2/GFP-expressing and nonexpressing cells confirmed a specific ablation of the colony-forming ability of GATA-2/GFP-expressing progenitor cells. Similarly, loss of spleen colony-forming ability was observed for GATA-2/GFP-expressing bone marrow cells. Despite enforced GATA-2 expression, marrow cells remained viable and were negative in assays to evaluate apoptosis. Although efficient transduction of primitive Sca-1+Lin- cells was observed with the GATA-2/GFP vector, GATA-2/GFP-expressing stem cells failed to substantially contribute to the multilineage hematopoietic reconstitution of transplanted mice. Additionally, mice transplanted with purified, GATA-2/GFP-expressing cells showed post-transplant cytopenias and decreased numbers of total and gene-modified bone marrow Sca-1+ Lin−cells. Although Sca-1+ Lin− bone marrow cells expressing the GATA-2/GFP vector were detected after transplantation, no appreciable expansion in their numbers occurred. In contrast, control GFP-expressing Sca-1+Lin− cells expanded at least 40-fold after transplantation. Thus, enforced expression of GATA-2 in pluripotent hematopoietic cells blocked both their amplification and differentiation. There appears to be a critical dose-dependent effect of GATA-2 on blood cell differentiation in that downregulation of GATA-2 expression is necessary for stem cells to contribute to hematopoiesis in vivo.

Kruppel-Like Factor 10 (KLF10)-Deficient Mice Have Marked Defects In EPC Differentiation, Function, and Angiogenesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4314-4314
Author(s):  
Akm Khyrul Wara ◽  
Kevin Croce ◽  
ShiYin Foo ◽  
Xinghui Sun ◽  
Basak Icli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Hindlimb Ischemia ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Deficient Mice ◽  
Tgf Β1 ◽  
Impaired Angiogenesis

Abstract Abstract 4314 Background: Emerging evidence demonstrates that endothelial progenitor cells (EPCs) may originate from the bone marrow and are capable of being recruited to sites of ischemic injury and contribute to neovascularization. However, the identities of these bone marrow cells and the signaling pathways that regulate their differentiation into functional EPCs remain poorly understood. Methods and Results: We previously identified that among hematopoietic progenitor stem cells, common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) can preferentially differentiate into EPCs and possess high angiogenic activity under ischemic conditions compared to megakaryocyte-erythrocyte progenitors (MEPs), hematopoietic stem cells (HSCs), and common lymphoid progenitors (CLPs). Herein, we identify that a TGF-β1-responsive Kruppel-like Factor, KLF10, is robustly expressed in EPCs derived from CMPs and GMPs, compared to progenitors lacking EPC markers. KLF10–/– mice have marked defects in circulating EPCs (–23.6% vs. WT, P&lt;0.004). In addition, EPC differentiation and TGF-β induced KDR responsiveness is markedly impaired (CMPs: WT 22.3% vs. KO 8.64%, P&lt;0.0001; GMPs: WT 32.8% vs. KO 8.97%, P&lt;0.00001). Functionally, KLF10–/– EPCs derived from CMPs and GMPs adhered less to fibronectin-coated plates (CMPs: WT 285 vs. KO 144.25, P&lt; 0.0004; GMPs: WT 275.25 vs. KO 108.75, P &lt;0.0003) and had decreased rates of migration in transwell Boyden chambers (CMPs: WT 692 vs. KO 298.66, P&lt;0.00004; GMPs: WT 635.66 vs. KO 263.66, P&lt;0.00001). KLF10–/– mice displayed impaired blood flow recovery after hindlimb ischemia (day 14, WT 0.827 vs. KO 0.640, P &lt;0.009), an effect completely rescued by WT EPCs, but not KLF10–/– EPCs. Matrigel plug implantation studies demonstrated impaired angiogenesis in KLF10–/– mice compared to WT mice (WT 158 vs. KO 39.83, P&lt;0.00000004). Overexpression studies revealed that KLF10 rescued EPC formation from TGF-β1+/– CMPs and GMPs. Mechanistically, TGF-β1 and KLF10 target the VEGFR2 promoter in EPCs which may underlie these effects. Background: Collectively, these observations identify that TGF-β1 signaling and KLF10 are part of a key signaling pathway that regulates EPC differentiation from CMPs and GMPs and may provide a therapeutic target during cardiovascular ischemic states. Disclosures: No relevant conflicts of interest to declare.

Non-Canonical NF-Kb Signaling Regulates Hematopoietic Stem Cell Self-Renewal and Microenvironment Interactions

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 859-859 ◽  
Author(s):  
Chen Zhao ◽  
Yan Xiu ◽  
John M Ashton ◽  
Lianping Xing ◽  
Yoshikazu Morita ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Double Knockout ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Physiological Processes ◽  
Marrow Cells

Abstract Abstract 859 RelB and NF-kB2 are the main effectors of NF-kB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-kB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-kB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Role Of Sf3b1 On Hematopoiesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 600-600
Author(s):  
Manabu Matsunawa ◽  
Ryo Yamamoto ◽  
Masashi Sanada ◽  
Aiko Sato ◽  
Yusuke Shiozawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Hematopoietic Stem Cells ◽  
Marrow Transplantation ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Ring Sideroblasts ◽  
Marrow Cells

Abstract Frequent pathway mutation involving multiple components of the RNA splicing machinery is a cardinal feature of myeloid neoplasms showing myeloid dysplasia, in which the major mutational targets include U2AF35, ZRSR2, SRSF2 and SF3B1. Among these, SF3B1 mutations were strongly associated with MDS subtypes characterized by increased ring sideroblasts, such as refractory anemia and refractory cytopenia with multiple lineage dysplasia with ring sideroblasts, suggesting the critical role of SF3B1 mutations in these MDS subtypes. However, currently, the molecular mechanism of SF3B1mutation leading to the ring sideroblasts formation and MDS remains unknown. The SF3B1 is a core component of the U2-small nuclear ribonucleoprotein (U2 snRNP), which recognizes the 3′ splice site at intron–exon junctions. It was demonstrated that Sf3b1 null mice were shown to be embryonic lethal, while Sf3b1 +/- mice exhibited various skeletal alterations that could be attributed to deregulation of Hox gene expression due to haploinsufficiency of Sf3b1. However, no detailed analysis of the functional role of Sf3b1 in hematopoietic system in these mice has been performed. So, to clarify the role of SF3B1 in hematopoiesis, we investigated the hematological phenotype of Sf3b1 +/- mice. There was no significant difference in peripheral blood counts, peripheral blood lineage distribution, bone marrow total cellularity or bone marrow lineage composition between Sf3b1 +/+ and Sf3b1 +/- mice. Morphologic abnormalities of bone marrow and increased ring sideroblasts were not observed. However, quantitative analysis of bone marrow cells from Sf3b1 +/- mice revealed a reduction of the number of hematopoietic stem cells (CD34 neg/low, cKit positive, Sca-1 positive, lineage-marker negative: CD34-KSL cells) measured by flow cytometry analysis, compared to Sf3b1 +/+ mice. Whereas examination of hematopoietic progenitor cells revealed a small decrease in KSL cell populations and megakaryocyte - erythroid progenitors (MEP) in Sf3b1 +/- mice, and common myeloid progenitors (CMP), granulocyte - monocyte progenitors (GMP) and common lymphoid progenitors (CLP) remained unchanged between Sf3b1 +/+ and Sf3b1 +/- mice. In accordance with the reduced number of hematopoietic stem cells in Sf3b1 +/- mice, the total number of colony-forming unit generated from equal number of whole bone marrow cells showed lower colony number in Sf3b1 +/- mice in vitro. Competitive whole bone marrow transplantation assay, which irradiated recipient mice were transplanted with donor whole bone marrow cells from Sf3b1 +/+ or Sf3b1 +/- mice with an equal number of competitor bone marrow cells, revealed impaired competitive whole bone marrow reconstitution capacity of Sf3b1 +/- mice in vivo. These data demonstrated Sf3b1 was required for hematopoietic stem cells maintenance. To further examine the function of hematopoietic stem cells in Sf3b1 +/- mice, we performed competitive transplantation of purified hematopoietic stem cells from Sf3b1 +/+ or Sf3b1 +/- mice into lethally irradiated mice together with competitor bone marrow cells. Sf3b1 +/- progenitors showed reduced hematopoietic stem cells reconstitution capacity compared to those from Sf3b1 +/+ mice. In serial transplantation experiments, progenitors from Sf3b1 +/- mice showed reduced repopulation ability in the primary bone marrow transplantation, which was even more pronounced after the second bone marrow transplantation. Taken together, these data demonstrate that Sf3b1 plays an important role in normal hematopoiesis by maintaining hematopoietic stem cell pool size and regulating hematopoietic stem cell function. To determine the molecular mechanism underlying the observed defect in hematopoietic stem cells of Sf3b1 +/- mice, we performed RNA-seq analysis. We will present the results of our biological assay and discuss the relation of Sf3b1 and hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Identification of a novel bone marrow-derived B-cell progenitor population that coexpresses B220 and Thy-1 and is highly enriched for Abelson leukemia virus targets.

1989 ◽  
Vol 9 (6) ◽  
pp. 2665-2671 ◽  
Author(s):  
G F Tidmarsh ◽  
S Heimfeld ◽  
C A Whitlock ◽  
I L Weissman ◽  
C E Müller-Sieburg
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Leukemia Virus ◽  
Cell Activity ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Low Levels ◽  
B Lineage ◽  
Marrow Cells

A novel stage in early B-lymphocyte differentiation has been identified in normal mouse bone marrow cells. Earlier work had demonstrated that bone marrow cells characterized by low levels of Thy-1 and lack of a panel of lineage markers (Thy-1lo Lin- cells) were highly enriched for pluripotent hematopoietic stem cells. In this paper, we present evidence that another bone marrow population, which expressed low levels of Thy-1 and coexpressed B220, a B-lineage-specific form of the leukocyte common antigen, contained early and potent precursors for B lymphocytes upon in vivo transfer to irradiated hosts. These Thy-1lo B220+ cells, comprising 1 to 2% of bone marrow cells, were enriched for large cells in the mitotic cycle; the population lacked significant pluripotent hematopoietic stem cell activity and myeloid-erythroid progenitors. Most strikingly, Thy-1lo B220+ cells represented a highly enriched population of bone marrow cells that could be targets of Abelson murine leukemia virus transformation. We propose that Thy-1lo B220+ bone marrow cells represent the earliest stage of committed lymphocyte progenitors, intermediate in differentiation between Thy-1lo Lin- pluripotent stem cells and, in the B lineage, Thy-1- B220+ pre-B cells.

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