scholarly journals Identification, Isolation and Transcriptome Analyses of Mouse, Rat and Man Erythroblastic Island Central Macrophages

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 841-841
Author(s):  
Wei Li ◽  
Yaomei Wang ◽  
Huizhi Zhao ◽  
Huan Zhang ◽  
Yuanlin Xu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Negative Control ◽  
Human Macrophage ◽  
Mouse Bone Marrow ◽  
Erythroid Cells ◽  
Wild Type ◽  
Erythroblastic Island ◽  
Hela Cell Lines

Abstract Erythroblastic island (EBI), composed of a central macrophage and surrounding erythroid cells, is the first hematopoietic niche discovered for erythropoiesis. Yet, the identity of the central macrophage has so far remained elusive. Based on the previous findings that F4/80, VCAM1 and CD169 are potential mouse central macrophage markers, we first calculated the number of F4/80+VCAM1+CD169+ mouse macrophages in the mouse bone marrow and compared it to the number of Ter119+ erythroblasts. We found that the ratio of F4/80+VCAM1+CD169+ macrophage and erythroblasts is about 1:2. Given the fact that one central macrophage is surrounded by multiple erythroblasts, the above finding suggests that it is unlikely that all the F4/80+VCAM1+CD169+ macrophages are central macrophages. Erythropoietin (Epo) is essential for erythropoiesis. It has been reported that the Epo receptor (Epor) is expressed in peritoneal macrophages. These findings promoted us to speculate that EBI central macrophages may express Epor so that Epo acts on both erythroid cells and the central macrophages simultaneously in the niche to ensure efficient and optimal red cell production. To test this notion, we first examined whether mouse bone marrow and fetal liver macrophages express Epor using the Epor-GFPcre knockin mouse model. We found that ~5% of bone marrow F4/80+ macrophages and ~35% of fetal liver F4/80+ macrophages express Epor-GFP. As negative control, no Epor-GFP macrophages are noted in wild type F4/80+ macrophages. Importantly, ImageStream analyses revealed the native EBIs in bone marrow and fetal liver are formed by Epor+ but not Epor- macrophages. Bioinformatics analyses of RNA-seq data on the sorted Epor+ and Epor- macrophage populations revealed that molecules involved in central macrophage-erythroblast association such as VCAM1, CD169, and molecules known to be important for central macrophage function such as Dnase2a, ferroportin, are highly expressed in Epor+ macrophages. In marked contrast, highly expressed pathways in Epor- macrophages are associated with immune responses including antigen process and presentation. Intriguingly, the immune related pathways are dramatically downregulated in the Epor+ macrophages, suggesting that the Epor+ macrophages in bone marrow and fetal liver have evolved a specialized function in supporting erythropoiesis. To examine whether expression of Epor in EBI central macrophages is a conserved feature across species, we generated Epor-GFPcre knockin rat using the CRISP/Cas9 technology. Using CD163 as rat macrophage marker, we found that a subpopulation of rat bone marrow CD163+ macrophages expresses Epor-GFP. As a negative control, no Epor-GFP macrophages are noted in wild type CD163+ macrophages. To examine whether EPOR is expressed in human EBI central macrophages, antibody specificity for human EPOR is critical. To this end, we employed CRISP/Cas9 approach to knock out EPOR in K562 and Hela cell lines and validated the specificity of a commercially available anti-human EPOR antibody. Using CD163, CD169 as human macrophage markers, we found that EPOR is also expressed in a subpopulation of human macrophages. Moreover, in vitro EBI formation assay revealed that human EPOR+ but not EPOR- macrophages form EBIs with erythroid cells and that the EBI formation is enhanced by EPO. In summary, we for the first time, after discovery of the EBIs 60 years ago, have identified Epor+ macrophages in mouse bone marrow and fetal liver as EBI central macrophages. Our findings provide solid foundation for studying the mechanisms by which erythropoieis is supported EBI central macrophages. A better understanding of such mechanisms will provide extensive new knowledge on basic biology of erythropoiesis. It is also important to understand the pathology of erythropoietic disorders as well as to improve ex vivo erythrocyte production. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Erythro-Myeloblastic Island (EMBI): A Hematopoietic Niche Balancing Erythropoiesis and Myelopoiesis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 842-842
Author(s):  
Katie Giger Seu ◽  
Laurel Romano ◽  
Julien Papoin ◽  
Edward David Muench ◽  
Diamantis Konstantinidis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Multispectral Imaging ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Mouse Bone Marrow ◽  
Erythroid Cells ◽  
Murine Models ◽  
Native Bone ◽  
Hematopoietic Niche

Abstract Mammalian erythropoiesis has long been established to occur within erythroblastic islands (EBIs), niches where erythroblasts differentiate in close contact with a central macrophage. While it is generally accepted that EBI macrophages play an important role in regulation of erythropoiesis, very little is known about the specific macrophage populations involved in EBI formation, the regulation that occurs within EBIs, or how this niche fits into the broader context of hematopoiesis. We analyzed native EBIs isolated from mouse bone marrow using multispectral imaging flow cytometry (Seu et. al. Front Immunol 2017). Consistent with historical observations, the EBIs were heterogeneous and many contained a number of closely CD11b+ cells in addition to erythroblasts and a central F4/80+ macrophage. Flow cytometry analysis of cells dissociated from native bone marrow EBIs indicated these niches are also enriched 2-3 fold in myeloblasts and granulocytic precursors up to metamyelocytes relative to the total bone marrow while they are depleted of mature granulocytes (bands and segmented cells). Bulk RNAseq of the CD11b+ population isolated from EBIs showed high expression of genes characteristic of the granulocytic lineage (e.g. Elane, Mpo, Gfi1, Cebpe, Camp, and Mmp9), indicating the EBI macrophages may regulate myelopoiesis along with erythropoiesis and that EBIs should really be considered as erythro-myeloblastic islands (EMBIs). To critically document the various hematopoietic cell populations that constitute EMBIs, we used the 10x Genomics Chromium system to obtain single cell gene expression data on ~3,500 total cells from isolated EMBIs along with at least 1,000 sorted cells from each of the 3 major EMBI-associated populations (F4/80+, CD71+, and CD11b+) (Fig 1a, b). The data were analyzed using 10x Genomics' Loupe cell Browser and Iterative Clustering and Guide-gene Selection (ICGS, http://www.AltAnalyze.org, Olsson et. al. Nature 2016). From the ICGS analysis, ~30% of the total EMBI-associated cells were myeloid cells that segregated into at least 3 transcriptionally distinct clusters representing granulocytic progenitors and precursors. As expected, erythroblasts with a progressive maturation pattern made up the bulk (60%) of the EMBI-associated cells, while up to 10% were a heterogeneous population of cells that exhibited expression of macrophage markers such as Csf1R and Irf8, along with genes previously described to characterize resident macrophages, such as Fn1and Fsp1/S100A4 (Fig 1c). In order to investigate the balance of myeloid cells with erythroid cells within the EMBIs, we examined the ratio of CD71+ cells to CD11b+ and how this ratio changes in models of altered granulopoiesis. While the number of myeloid cells at any island varied, the overall ratio of CD11b+ area to CD71+ within the EMBIs was relatively constant at steady state. In three different murine models of anemia of inflammation (AoI), we found that this ratio of CD11b+ to CD71+ cells within the EMBI increases dramatically indicating that the increased granulopoiesis and suppression of erythropoiesis noted in AoI is a result of altered balance of the hematopoiesis within the EMBI unit. Similarly, stimulation of granulopoiesis with GCSF also results in a shift within the EMBIs to CD11b+ myeloid cells and suppression of erythroid cells. Alternatively, in gfi1 KO mice, a model of congenital neutropenia in which granulopoiesis fails at an early stage, the ratio shifts toward CD71+ erythroid cells with paucity of the granulocytic precursors that are typically found at the EMBIs. Taken together, these data indicate that granulocyte progenitors and precursors are specifically associated with EMBI macrophages in the mouse bone marrow. The preferential localization of myeloid precursors within EMBIs suggests this niche is a site for granulopoiesis as well as erythropoiesis and production of these lineages is dynamically regulated within this niche. Our work with multiple murine models of altered granulopoiesis demonstrates that pathological expansion of one of the lineages within this niche may suppress the other and that the interactions within the EMBI could be a useful therapeutic target for AoI. These novel findings significantly broaden our understanding of the role of this hematopoietic niche in the regulated development of lineage committed erythroid and myeloid cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Ap2a2 Is Crucial for LT-HSC Quiescence

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2642-2642
Author(s):  
Stephen B Ting ◽  
Sara Rhost ◽  
Sarah Ghotb
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Fold Increase ◽  
Wild Type ◽  
Lacz Reporter ◽  
Adult Survivor ◽  
Self Renewal ◽  
Post Transplant

Abstract Despite the relative rarity of haematopoietic stem cells (HSCs) within the blood system, functional heterogeneity is paramount to their ability to sustain lifelong blood production. The quiescent HSC sits at the functional apex possessed with self-renewal properties and the greatest repopulation output. We previously identified the gene, Ap2a2 as an enhancer of HSC function and its protein as a potential cell fate determinant in HSC asymmetric cell divisions (Ting SB et al., Blood 2012). Mechanistically, we hypothesise Ap2a2 induces a state of HSC quiescence. Using the Tet-On histone H2B-GFP mouse model (Foudi et al., Nat Biotech 2009), we have shown Ap2a2 to be highly and differentially expressed in the predominantly, G0 dormant CD150+48-LSK GFPhigh as opposed to the more cycling GFPlow HSC subpopulation. Competitive transplantation of Ap2a2- versus empty vector-transduced H2B-GFP HSCs results in a three-fold increase of the CD150+48-LSK GFPhigh HSC subpopulation. To further confirm the importance of Ap2a2 in haematopoiesis, we have constructed Ap2a2-LacZ reporter and constitutive Ap2a2 knockout (KO) mouse lines. The Ap2a2 LacZ reporter with b-galactosidase flow cytometry staining of bone marrow subpopulations confirmed high endogenous Ap2a2 expression in the CD150+48-LSK long-term (LT-) versus CD150-48-LSK short-term (ST-) repopulating HSCs. Interim analyses of the constitutive Ap2a2 KO mice have revealed two obvious phenotypes: 14% of Ap2a2-null mice termed "non-survivors" are smaller, paler with failure of fetal liver (FL) development and die between E18.5 and weaning, whilst the remaining 11% are adult viable "survivors". However, at E14.5, Ap2a2-null compared to Ap2a2-wild type fetal livers showed less absolute total FL cells but increased CD150+48-LSM FL HSCs. This was quantitatively correlated via limiting dilution assay assessed at 16 weeks post-transplant with a two-fold increase in Ap2a2-null HSC numbers (1 in 78,917 versus 1 in 150,891, p=0.027). This suggests Ap2a2 has a role in FL HSC differentiation and/or fate with potential impairment of symmetrical versus asymmetrical HSC divisions currently being studied. When E14.5 FL cells were competitively transplanted, the Ap2a2-null HSC had impaired donor reconstitution function measured at 16 weeks post-transplant (19.8% versus 48.6%, p=0.015). Ap2a2-null versus wild-type E14.5 FL cells showed equivalent numbers of primary in vitro methylcellulose colony assays but loss of secondary colonies upon re-plating indicative of loss of in-vitro HSC self-renewal. Importantly, although the Ap2a2 adult "survivors" exhibited normal quantities of bone marrow HSC subpopulations, when functionally assessed, Ap2a2-null adult "survivor" HSCs showed loss of in-vivo HSC self-renewal in secondary transplantation assays. To investigate potential cellular mechanisms, we studied the cell cycle state of Ap2a2-null and wild-type E14.5 FL cells and identified that Ap2a2-null "non-survivors" had a relative loss of quiescent G0, specifically in the LT-HSC (and not seen in the ST-HSC) subpopulation throughout all of (E14.5 to E18.5) FL development. In contrast, the LT-HSC subpopulation in FLs of Ap2a2-null "survivors" had an initial loss of G0 at E14.5 but a compensatory increase in LT-HSC G0 by E18.5. Our preliminary data suggests Ap2a2 is a crucial factor for the quiescent LT-HSC subpopulation, and we propose that both during the highly proliferative fetal liver stage of haematopoiesis and adult HSCs under stress that Ap2a2 maintains a critical balance of dormant ("deep-sleeper") HSCs to ensure global HSC function. Disclosures No relevant conflicts of interest to declare.

scholarly journals Radiosensitivity of Fancd2-/- mouse Bone Marrow Stromal Cells Is Altered By Abrogation of TGF-β Signaling

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4796-4796
Author(s):  
Katherine Chen ◽  
Darcy Franicola ◽  
Donna Shields ◽  
Michael W. Epperly ◽  
Xichen Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Survival Curve ◽  
Radiation Sensitivity ◽  
Mouse Bone Marrow ◽  
Wild Type ◽  
Increased Risk ◽  
Significant Difference

Abstract Both marrow-transplanted and non-transplanted Fanconi Anemia (FA) patients are often radiosensitive. Due to an increased risk of developing secondary malignancies, these patients require dose and volume modification during radiotherapy. To determine whether abrogation of TGF-β signaling alters the radiation sensitivity of Fancd2-/- mice, cell lines derived from double knockout (DKO) (SMAD3-/- Fancd2-/-) mice were compared with those from Fancd2-/-, SMAD3-/-, and wild-type mice for ionizing irradiation sensitivity. Bone marrow stromal cell lines were derived from long-term bone marrow cultures of DKO, Fancd2-/-, SMAD3-/-, and wild-type SMAD3+/+ (129/Sv) X Fancd2+/+ (B6) F1 mice. Radiation sensitivity was determined using clonogenic irradiation survival curves. There was no significant difference in radiosensitivity comparing DKO cells (Do = 1.95 ± 0.06 Gy, ň = 4.3 ± 0.7) to the wild type SMAD3+/+ (129/Sv) X Fancd2+/+ (B6) F1 cell line (Do = 2.00 ± 0.11 Gy, and ň = 5.1 ± 0.7, p = 0.7003 and 0.4820, respectively). The Fancd2-/- cell line was more radiosensitive with a Do of 1.37 ± 0.09 Gy compared to 1.95 ± 0.07 and 2.00 ± 0.11 for DKO and wild type cells (p = 0.0063 and 0.0360, respectively. In contrast, the SMAD3-/- cell line was more radioresistant with an increased shoulder on the irradiation survival curve (ň = 12.1 ± 2.9) compared to the DKO or wild type SMAD3+/+ (129/Sv) X Fancd2+/+ (B6) F1 cell lines (ň = 4.335 ± 0.7 or 5.1 ± 0.7, p = 0.00277 or 0.0426, respectively). This confirms and extends results with SMAD3-/- mouse derived cell lines on another background strain (C57BL/6J) (Epperly, et al., Radiation Research, 165:671-677, 2006). TGF-β signaling was abrogated in both DKO and SMAD3-/- mouse cell lines (measured by TGF-β inhibition of fresh marrow CFU-GEMM in vitro), confirming the phenotype of altered TGF-β signaling. Therefore, radiosensitivity associated with the Fancd2-/- genotype was abrogated by interruption of the TGF-β signaling pathway in the same cells. Supported by research grant NIAID/NIH, U19A168021. Disclosures No relevant conflicts of interest to declare.

CSF3R T618I Mouse Bone Marrow Transplant Model Of Neutrophilic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 223-223
Author(s):  
Julia E Maxson ◽  
Angela G. Fleischman ◽  
Samuel B Luty ◽  
Lacey R Royer ◽  
Anupriya Agarwal ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Clinical Trials ◽  
Bone Marrow Transplant ◽  
Conflicts Of Interest ◽  
Marrow Transplant ◽  
Mouse Bone Marrow ◽  
Wild Type ◽  
Cell Counts ◽  
Financial Interest ◽  
White Blood Cell Counts

Abstract Background We have recently identified mutations in CSF3R in the majority of patients with chronic neutrophilic leukemia (CNL). The most common mutation identified thus far is the T618I membrane proximal mutation, which confers rapid transformation capacity and ligand-independence to the receptor. We also observed less common mutations that result in truncation of the cytoplasmic domain of CSF3R. These truncation mutations, which have been studied in the context of severe congenital neutropenia progression to acute myeloid leukemia, lack the capacity for inducing disease in transgenic mouse models but enhance the progression to leukemia in the presence of a second genetic driver. The CSF3R T618I mutation transforms cells at a substantially faster rate than the truncation mutation, but the capacity of this mutation to induce a hematologic disorder in mice is not yet known. We created a CSF3R T618I mouse bone marrow transplant model, which resulted in a lethal myeloproliferative disorder characterized by high levels of granulocytes. Methods Mice were transplanted with donor bone marrow infected with a murine retrovirus expressing either wild type CSF3R or the CSF3R T618I mutation. White blood cell counts were measured 1-2 times per week for 90 days. The percentages of monocytes, granulocytes, T-cells and B-cells were analyzed by flow cytometery. The levels of pSTAT3 were measured by phospho-flow. Hematoxylin and eosin staining was performed on fixed and sectioned spleens, livers and bone marrow. Results Mice transplanted with CSF3R T618I showed an initial transient granulocytic leukocytosis that normalized by 33 days post transplant. Subsequently, at day 47 the CSF3R T618I mice exhibited a dramatic and persistent rise in white blood cell counts. The elevated white blood cells were primarily mature granulocytes. By day 90, all of the mice transplanted with CSF3R T618I had died, while the mice transplanted with cells expressing wild type CSF3R did not exhibit morbidity or mortality. The granulocytes from CSF3R T618I mice also exhibited elevated levels of pSTAT3—a marker of JAK signaling downstream of CSF3R—relative to CSF3R wild type granulocytes. Histology revealed that both CSF3R wild type and T618I mice had hypercellular bone marrow, however the hypercellularity was more extreme in the CSF3R T618I mice. Mice harboring the CSF3R T618I and to a lesser extent wild type CSF3R exhibited infiltration of the spleen and liver by mature granulocytic cells and disrupted tissue architecture. Conclusion The CSF3R T618I mouse bone marrow transplant provides a tractable model of Neutrophilic Leukemia. In contrast to CSF3R truncation mutations, which do not cause leukemia in isolation, the T618I mutation causes a fatal expansion of granulocytes. This model provides a useful tool for studying the biology of granulocytes and myeloproliferative neoplasms. Disclosures: Off Label Use: Ruxolitinib - a JAK1/2 inhibitor that we propose can be used off-label for disease management of CSF3R-mutant neutrophilic leukemia. Fleischman:Incyte: Speakers Bureau. Druker:Incyte: PI or co-investigator on clinical trials., PI or co-investigator on clinical trials. Other; Novartis: PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU., PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other; Bristol-Myers Squibb: PI or co-investigator on BMS clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other. Tyner:Incyte Corporation: Research Funding.

Targeted Gene Deletion Demonstrates That Adhesion Molecule ICAM-4 Is Critical for Erythroblastic Island Formation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1661-1661
Author(s):  
Gloria Lee ◽  
Annie Lo ◽  
Sarah Short ◽  
Tosti Mankelow ◽  
Stephen Parsons ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Intracellular Signaling ◽  
Marked Decrease ◽  
Immunoglobulin Superfamily ◽  
Mouse Bone Marrow ◽  
Wild Type ◽  
Erythroid Progenitors ◽  
Island Formation ◽  
Erythroblastic Island

Abstract Erythroid progenitors proliferate, differentiate and enucleate within specialized bone marrow subcompartments, termed erythroblastic islands, which are comprised of developing erythroblasts surrounding a central macrophage. Growing evidence suggests that within erythroblastic islands adhesion events, in concert with cytokines, play critical roles in regulating erythropoiesis and apoptosis. We are exploring the potential function of erythroid ICAM-4, a recently characterized member of the immunoglobulin superfamily, in erythroblastic island formation. We earlier identified α4β1 and αV integrins as ICAM-4 binding partners. Since erythroblasts express α4β1 and ICAM-4 and macrophages exhibit αV, ICAM-4 is an attractive candidate for mediating erythroblast-erythroblast and erythroblast-macrophage attachments. Indeed, two synthetic peptides that block ICAM-4/αV adhesion caused a marked decrease in the percentage of islands formed. To more definitively test whether ICAM-4 attachments are active in erythroblastic islands we generated ICAM-4 knockout mice and compared the capacity of single cell suspensions from freshly harvested ICAM-4 null and wild type bone marrow to form erythroblastic islands in vitro, using a reproducible live cell island reconstitution assay that we have established. Islands and their cellular components were identified and quantitated by three-color immunofluorescent microscopy employing fluoresceinated erythroid-specific TER119 antibody, macrophage-specific F4/80 antibody and a DNA probe. Strikingly, we observed a 47% decrease in the percentage of islands formed from bone marrow of ICAM-4 null mice compared to wild type littermates (n=10 and n=10, respectively). We also studied the ability of ICAM-4 null erythroblasts to form islands in vivo by analyzing intact islands freshly harvested from mouse bone marrow. Similar to the in vitro data we found a marked decrease in the percentage of islands formed in the bone marrow of ICAM-4 null mice compared to wild type littermates. The null mice had 44% fewer islands than wild type mice. Taken together, the results of this phenotypic analysis provide convincing evidence that ICAM-4 is one of the adhesion molecules critical for erythroblastic island formation. We postulate that this newly identified erythroblast receptor may be important not only for adhesive integrity of the island structure but also for initiating intracellular signaling essential for normal erythroid terminal differentiation.

Increased Interferon-αa Signaling During Megakaryocyte Ontogeny: Implications for the Spontaneous Resolution of Down Syndrome Transient Myeloproliferative Disorder.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1272-1272 ◽  
Author(s):  
Karen Wieland ◽  
Andrew Woo ◽  
Thomas Akie ◽  
Alan B. Cantor
Keyword(s):  
Bone Marrow ◽  
Down Syndrome ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Myeloproliferative Disorder ◽  
Spontaneous Resolution ◽  
Chromosome 21 ◽  
Dependent Manner ◽  
Wild Type ◽  
Transient Myeloproliferative Disorder

Abstract Abstract 1272 Poster Board I-294 About ten percent of infants with Down syndrome (DS) are born with a transient myeloproliferative disorder (DS-TMD), which spontaneously resolves within the first few months of life. However, the basis for this resolution remains unknown. Acquired mutations leading to exclusive production of a short isoform of the transcription factor GATA-1 (GATA-1s) occur in all cases of DS-TMD, and knock-in mice that exclusively produce GATA-1s have hyperproliferation of megakaryocytes during early fetal liver hematopoiesis, but not during later developmental stages. In this study, we found striking upregulation of the interferon-αa (IFN-αa) receptor and multiple IFN-αa responsive genes, including Ifi203, Ifi205, Irf-1, Irf-8, and Ifitm6, in immunophenotypically isolated megakaryocyte progenitor cells (MkPs) from bone marrow versus embryonic day 13.5 (e13.5) fetal liver of wild type mice. These differences were confirmed at the protein level in megakaryocytes by in situ immunohistochemistry. Addition of IFN-αa to GATA-1s containing e13.5 fetal liver MkPs reduces their hyperproliferation in vitro in a dose-dependent manner. Conversely, injection of neutralizing IFN-αa/β antibodies, but not control IgG, into adult GATA-1s mice markedly increases the percentage of bone marrow CD41+ cells and morphologically recognizable megakaryocytes, in contrast to wild type mice. We propose that increases in IFN-αa signaling during megakaryocyte ontogeny may account for the developmental stage-specific effects of GATA-1s on megakaryocyte hyperproliferation, and possibly the spontaneous resolution of DS-TMD. Interestingly, the genes encoding the IFN-αa/β receptor are located on human chromosome 21 and are expressed at 1.6 times that in trisomy versus disomy 21 cells. We speculate that increased interferon alpha signaling during embryogenesis may be the basis for the strong selective pressure for GATA-1s producing mutations in trisomy 21 fetuses in the first place. Disclosures No relevant conflicts of interest to declare.

Fetal Vs. Adult Hematopoietic Progenitors Respond Differently to NOTCH1: Implications for Pediatric Vs. Adult T-ALL.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2381-2381
Author(s):  
Vincenzo Giambra ◽  
Sonya H Lam ◽  
Amy Ng ◽  
Claudia Benz ◽  
Olena O Shevchuk ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Lymphoblastic Leukemia ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Adult Bone Marrow ◽  
Old Adult ◽  
Adult Bone

Abstract Abstract 2381 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of T-cell progenitors which affects both children and adults. Whereas pediatric T-ALL is curable in 80–90% of cases, only 40% of adults with T-ALL survive beyond 5 years. Fetal liver and adult bone marrow-derived hematopoietic stem/progenitor cells (HSPCs) are known to differ in terms of their gene expression programs and functional properties. Despite this work, the extent to which differential programming of fetal and adult HSPCs may impact the biology of their respective leukemias in children and adults remains unexplored. NOTCH1 is a prominent oncogene in T-ALL and activated by mutation in over 50% of cases. The retroviral NOTCH1 mouse bone marrow transplant model of T-ALL is well established; however, most investigators use 8–12 week-old adult mice as bone marrow donors for these experiments and thus these studies could be interpreted as having modeled adult disease. In order to explore the possibility that fetal program HSPCs would more accurately model pediatric disease, we transduced lineage-negative fetal liver HSPCs with activated NOTCH1 (ΔE) retrovirus, transplanted them into syngeneic (C57BL/6) recipients, and compared the behavior of the resulting leukemias to those generated from lineage-negative 8-week-old adult bone marrow HSPCs. Primary transplant recipient mice developed nearly identical T-ALL disease in terms of penetrance, latency, disease distribution/burden, and immunophenotype. Serial transplantation of these leukemias into secondary recipients, however, revealed stark differences in that whereas “adult” leukemias were readily transplantable, “fetal” leukemias were largely non-transplantable. In order to quantitate leukemia-initiating cell (LIC) frequencies in these two situations, we performed secondary transplants into highly permissive, immunodeficient (NOD/Scid/Il2rg−/−) recipients at limiting dilution and observed fetal leukemias to exhibit 500-fold lower LIC activity than adult leukemias (1 in ∼4500 cells vs. 1 in ∼9 cells, respectively). To identify potential mechanisms that might underlie this difference in LIC activity, we compared the behaviors of fetal liver vs. adult bone marrow-derived HSPCs shortly after transduction with NOTCH1(ΔE) virus. Interestingly, NOTCH1 induced fetal HSPCs to cycle rapidly whereas adult HSPCs were largely quiescent. We also noted that non-transduced cells in fetal HSPC cultures were also cycling rapidly, and through a series of fetal/adult mixing and conditioned media experiments, we determined that NOTCH1 induces an autocrine IGF1 signaling circuit in fetal, but not adult HSPCs. This observation was also confirmed to hold true for CD34+ human cord blood vs. adult bone marrow HSPCs. Expression profiling/qRT-PCR and chromatin immunoprecipitation (ChIP) studies further revealed NOTCH1 to induce IGF1 transcription and altered chromatin structure (increased H3K4me3 and decreased H3K27me3 marks) specifically in fetal, but not adult HSPCs. These findings suggest that developmental stage-specific programming in fetal vs. adult progenitors underlies their differential responses to oncogenic NOTCH1 signaling, and also the biological aggressiveness of resulting leukemias. Therapeutic targeting of adult-specific pathways may thus achieve improved clinical responses in adults with T-ALL and perhaps also the minority of pediatric patients with more aggressive, possibly “adult-like” disease. Disclosures: No relevant conflicts of interest to declare.

Erythropoietin-Driven Signaling Complements the Survival Defect of DMT1-Mutant Erythroid Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2104-2104
Author(s):  
Monika Horvathova ◽  
Katarina Kapralova ◽  
Zuzana Zidova ◽  
Dalibor Dolezal ◽  
Dagmar Pospisilova ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Post Treatment ◽  
High Dose ◽  
Erythroid Cells ◽  
Wild Type ◽  
Normal Range ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Pre Treatment

Abstract Abstract 2104 Mutations inactivating the divalent metal transporter 1 (DMT1) cause impaired erythroid iron utilization and lead to the development of hypochromic microcytic anemia associated with ineffective erythropoiesis (IE). The anemia can be ameliorated with high-dose erythropoietin (EPO) therapy (Pospisilova D, et al. Blood. 2006. 108:404–5). In contrast to β-thalassemia mouse model with dramatically elevated EPO levels which were proposed to protect erythroid cells from apoptosis (Libani IV, et al. Blood. 2008. 112:875–885), DMT1-mutant mice (mk/mk) have only 2.8-fold higher EPO levels when compared to the wild-type littermates. This corresponds to 2-fold elevation of serum EPO above the normal range for DMT-1-mutant patient before initiation of EPO therapy. Different mechanisms may therefore drive IE in anemia due to DMT1 mutation. In this study we analyzed the bases for the clinical success of high-dose EPO supplementation in a DMT1-mutant patient and mk/mk mice. EPO administration significantly increased hemoglobin levels (7.4 g/dL to 9.1 g/dL for the patient and 7.5±0.6 to 9.5±0.4 g/dL for mk/mk mice) and partially ameliorated IE. Colony forming assay using patient's cells showed significantly improved in vitro growth of post-treatment DMT1-mutant burst-forming unit erythroid (BFU-E) progenitors when compared to pre-treatment BFU-Es. In addition, the reduced plating efficiency and colony-forming capacity of pre-treatment DMT1-mutant BFU-Es can be corrected by the addition of the broad spectrum caspase inhibitor z-VAD-fmk to the cultures. This indicates involvement of caspase-dependent apoptosis in the defective survival of pre-treatment BFU-E progenitors and in their impaired capacity to form erythroid colonies. TUNEL assay on patient's bone marrow smears showed markedly decreased rate of apoptosis (from 4% to 1.5% of TUNEL-positive erythroblasts) after EPO supplementation. No profound changes in erythroblast maturation were noted in post-treatment bone marrow with the exception of additional expansion of polychromatophilic pool suggesting that inhibition of apoptosis rather than increased differentiation of DMT1-mutant erythroid cells predominantly accounts for amelioration of anemia and IE. In accordance with the patient's results, EPO administration to mk/mk mice did not alter the distribution of erythroblasts of different maturation stages. On the other hand, augmented STAT5 activation and enhanced expression of anti-apoptotic proteins BCL-XL and MCL-1 was detected in EPO treated mice. This correlated with decreased number of erythroid Ter119+ precursors undergoing apoptosis in EPO treated mk/mk bone marrow (12.4±2.3% to 5.4±0.9%) and spleen (7.3±0.7% to 3.1±0.9%). EPO supplementation also significantly reduced susceptibility of mk/mk erythrocytes to undergo stress-induced death that could reflect increased eryptosis (apoptosis of DMT1-mutant erythrocytes) in vivo and protective effect of EPO. Low to undetectable expression of hepcidin in mk/mk liver could be attributed to 16-fold increase in GDF15 expression in the bone marrow; the expression of TWSG1 was comparable to wild-type littermates. Also patient's urinary hepcidin is low (55.3 ng/mg creatinine; normal range 71–1762), however, in contrast to mk/mk mice and β-thalassemia patients the suppression of hepcidin seems to be only partly mediated by GDF15 as patient's GDF15 plasma levels are only 1.9-fold higher (548.4 pg/mL) in comparison to gender- and age-matched controls (288.4±56.9 pg/mL). These results indicate that mouse models may not fully mimic the human disease and suggest existence of additional bone marrow-derived regulator of hepcidin expression. In summary we present the bases for the clinically approved success of EPO treatment under condition of iron-deprived erythropoiesis. We conclude that EPO-driven signaling rescues the survival defect of DMT1-mutant erythroid cells. Grant support: Czech Grant Agency, grants No. P305/10/P210 and P305/11/1745; Internal Grant of Palacky University Olomouc (LF_2011_011), and Ministry of Health Czech Republic Grant NS10281-3/2009. Disclosures: No relevant conflicts of interest to declare.

STAT3 Is Differentially Activated Downstream of the Erythropoietin Receptor in Primitive Compared to Definitive Erythroblasts

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2383-2383
Author(s):  
Jeffrey Malik ◽  
Samantha J England ◽  
Paul D Kingsley ◽  
James Palis
Keyword(s):  
Target Genes ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Erythropoietin Receptor ◽  
Erythroid Cells ◽  
Wild Type ◽  
Stat3 Signaling ◽  
Stat3 Phosphorylation

Abstract Abstract 2383 Erythropoietin (EPO) acting through its receptor (EPOR), is a critical regulator of definitive erythropoiesis. The EPOR signaling network has been extensively studied in cell lines overexpressing various forms of EPOR. Together with genetic models, these studies have defined an EPOR-JAK2-STAT5 pathway that is critical for the formation of red blood cells. However, the hematopoietic phenotype of mice with a truncated EPOR incapable of activating STAT5 (EPOR-HM) suggests that STAT5 activation is not the only pathway mediating the erythropoietic effects of EPO-EPOR signaling. Our recent studies indicate that EPO-EPOR signaling also regulates primitive erythroblast maturation in the mammalian embryo. To better understand EPOR signaling in the context of primary erythroid cells, we utilized primitive erythroblasts from wild-type and EPOR-HM murine embryos. Robust phosphorylation of STAT5 was detected by western blot in freshly isolated primitive erythroblasts from wild-type embryos. As expected, there is no STAT5 phosphorylation evident in primitive erythroblasts from EPOR-HM embryos. Surprisingly, EPOR-HM embryos produce nearly normal numbers of primitive erythroblasts and we detected equivalent induction of STAT target genes following EPO stimulation in wild-type and EPOR-HM primitive erythroblasts. To address the discrepancy between these data and the accepted model of EPOR-STAT5 signaling, we searched for compensatory signaling via other STAT family members. We found that STAT3, but not STAT1, was phosphorylated in primitive erythroblasts from EPOR-HM embryos, suggesting that phospho-STAT3 compensates for the loss of phospho-STAT5 in the EPOR-HM mice. We also detected a lower level of STAT3 phosphorylation in wild-type primitive erythroblasts that was increased upon in vitro EPO stimulation. These results indicate that EPOR-STAT3 signaling normally occurs in primitive erythroblasts. Furthermore, our preliminary data show that STAT3 transcript levels are higher in primitive erythroblasts compared to definitive erythroblasts from the fetal liver or bone marrow, suggesting that STAT3 activation during erythropoiesis may be lineage-dependent. To test this hypothesis, we utilized a small molecule inhibitor of STAT3 to examine the function of STAT3 signaling both in primitive and in definitive erythroblasts during in vitro maturation. The terminal maturation of definitive erythroblasts is unaffected by STAT3 inhibition. However, inhibition of STAT3 causes a 40% reduction in primitive erythroblast proliferation in a novel 2-step ex vivo culture system. We conclude that, in contrast to definitive erythroblasts, STAT3 is activated downstream of EPOR in primitive erythroid cells. EPOR-STAT3 signaling has been reported in non-erythroid cells as well as in several cancer cell types. Therefore the study of EPOR signaling in primary embryonic erythroblasts may provide novel insights regarding the function of EPO in these cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Inducible production of human macrophage growth factor, CSF-1

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 633-639 ◽  
Author(s):  
P Ralph ◽  
MK Warren ◽  
MT Lee ◽  
J Csejtey ◽  
JF Weaver ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Bone Marrow Cells ◽  
Colony Growth ◽  
Northern Analysis ◽  
Human Macrophage ◽  
Nonspecific Esterase ◽  
Mouse Bone Marrow ◽  
Serum Free ◽  
Murine Bone Marrow

A panel of human cell lines was screened for production of colony- stimulating factor-1 (CSF-1) using a specific radioreceptor assay and criterion of macrophage colony growth in mouse bone marrow culture. The pancreatic carcinoma lines MIA PaCa and PANC were found to secrete high levels of CSF-1. In a bone marrow proliferation assay, the activities from these two lines were blocked by a CSF-1 specific neutralizing antiserum, confirming the predominant content of this macrophage growth factor. MIA PaCA cells stopped secreting CSF-1 when transferred to various serum-free media. Serum-free production could be reinitiated by phorbol myristic acetate (PMA). Purified CSF-1 from serum-free MIA PaCa cells stimulated the formation of 14-day colonies from total and nonadherent mononuclear human bone marrow cells. Most of the colonies consisted exclusively of large, dispersed macrophages that were intensely stained for nonspecific esterase. Although similar numbers of human 14-day colonies were stimulated by CSF-1 and other CSFs, more CSF- 1 was required for the proliferation of human as compared with murine bone marrow progenitors. Northern analysis of mRNA from induced-MIA PaCa cells, using a human CSF-1 oligonucleotide probe, revealed multiple species of CSF-1 mRNA ranging from 1.5 to 4.5 kilobases (kb). Uninduced, serum-free cultures showed only the largest mRNA species, suggesting that serum removal interfered with CSF-1 mRNA processing related to synthesis and/or secretion of the protein. Regulation of the production of CSF-1 may be an important physiological process in hematopoiesis and macrophage functioning.

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