Transcriptome- Based Analysis of EPO Dependent Cfu-e to Erythroblast Development

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3188-3188
Author(s):  
Rakesh Verma ◽  
Aishwarya Narayanan ◽  
David Kuhrt ◽  
Lei Li ◽  
Su Su ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Murine Bone Marrow ◽  
Correlation Score ◽  
Insight Into

Abstract Abstract 3188 To provide molecular insight into erythroid developmental programs, including EPO- regulated aspects, we have employed transcriptome-based approaches to analyze the stage-wise development of purified murine bone marrow- derived CFUe, proerythroblasts and maturing erythroblasts. In vivo and ex vivo, these progenitors develop as KitposCD71highTer119neg; KitnegCD71highTer119neg; and KitnegCD71highTer119pos cohorts (designated as E1, E2, E3 stages, respectively). In the context of EPO- modulation, stage E1 cells exhibited ∼250 EPO- regulated target genes. In stage-E2 proerythroblasts, in contrast, 750 transcripts proved to be EPO- regulated while stage-E3 erythroblasts exhibited only select EPO- modulated genes (<50). At E1 and E2 stages, EPO- regulated targets included overlapping yet distinct sets, and this was reflected in functional sets of GO terms. Major EPO- targets at stage E1 included cell cycle and cell biogenesis genes, while in E2 proerythroblasts, negative regulators of protein kinases (and kinase activity) constituted a major EPO/EPOR target set. As E1 CFUe transitioned to E2 proerythroblasts, an unexpected transient narrowing in the complexity of overall expressed genes was exhibited. Stage- modulated global sets of transcripts included myeloid cell differentiation factors, cell number homeostasis factors and heme biosynthetic processes. As E2 proerythroblasts transitioned to E3 erythroblasts, functional GO sets were associated predominantly with dynamics in organelle and cellular compartments. In addition, HomoloGene and Connectivity Mapping approaches were applied to compare transcriptomes of stage E1, E2 and E3 murine bone marrow erythroid progenitors with four recently studied stages of human erythroid progenitor cell development (here, termed H1, H2, H3 and H4). High correlation of stage E1 m-CFUe with not only human H1 CFUe but also H2 proerythroblasts was observed (0.59 and 0.57 correlations). Stage E2 murine proerythroblasts best corresponded to H2 human proerythroblasts (0.36 correlation score), while E3 murine erythroblasts aligned closely with human H4 late stage erythroblasts (0.58 correlation score). These latter analyses provide novel transcriptome- based comparisons, and transcript specific insight, into conserved vs distinct features of murine and human erythroid development at CFUe to maturing erythroblast stages. Disclosures: No relevant conflicts of interest to declare.

Abnormal Distribution of Erythroid Progenitors Populations in Mice Lacking p45 NF-E2.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1988-1988
Author(s):  
Jadwiga Gasiorek ◽  
Gregory Chevillard ◽  
Zaynab Nouhi ◽  
Volker Blank
Keyword(s):  
Bone Marrow ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Globin Genes ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Adult Mice ◽  
Deficient Mice

Abstract Abstract 1988 Poster Board I-1010 The NF-E2 transcription factor is a heterodimer composed of a large hematopoietic-specific subunit called p45 and widely expressed 18 to 20-kDa small Maf subunits. In MEL (mouse erythroleukemia) cells, a model of erythroid differentiatin, the absence of p45 is inhibiting chemically induced differentiation, including induction of globin genes. In vivo, p45 knockout mice were reported to show splenomegaly, severe thrompocytopenia and mild erythroid abnormalities. Most of the mice die shortly after birth due to haemorrhages. The animals that survive display increased bone, especially in bony sites of hematopoiesis. We confirmed that femurs of p45 deficient mice are filled with bone, thus limiting the space for cells. Hence, we observed a decrease in the number of hematopoietic cells in the bone marrow of 3 months old mice. In order to analyze erythroid progenitor populations we performed flow cytometry using the markers Ter119 and CD71. We found that p45 deficient mice have an increased proportion of early erythroid progenitors (proerythroblasts) and a decreased proportion of late stage differentiated red blood cells (orthochromatic erythroblasts and reticulocytes) in the spleen, when compared to wild-type mice. We showed that the liver of p45 knockout adult mice is also becoming a site of red blood cell production. The use of secondary sites, such as the spleen and liver, suggests stress erythropoiesis, likely compensating for the decreased production of red blood cells in bone marrow. In accordance with those observations, we observed about 2 fold increased levels of erythropoietin in the serum of p45 knockout mice.Overall, our data suggest that p45 NF-E2 is required for proper functioning of the erythroid compartment in vivo. Disclosures: No relevant conflicts of interest to declare.

The Erythropoiesis Stimulating Agent Omontys/Peginesatide up-Modulates Erythroid Progenitor Formation, and Cell Surface Erythropoietin Receptor Levels.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2089-2089
Author(s):  
Rakesh Verma ◽  
Jennifer M Green ◽  
Karen Leu ◽  
Richard B Mortensen ◽  
Peter R. Young ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Surface ◽  
Ex Vivo ◽  
Erythropoietin Receptor ◽  
Cell Surface Expression ◽  
Dialysis Patients ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Murine Bone Marrow ◽  
Erythroid Development

Abstract Abstract 2089 Peginesatide is a peptide-based erythropoietin receptor (EPOR) agonist with recent FDA approval for treating the anemia of chronic kidney disease among adult dialysis patients. Although peginesatide exhibits a 47.9h half-life when given IV in dialysis patients, it is administered once-monthly. Taken together, this predicts that additional functional properties contribute to peginesatide's durable erythropoiesis stimulating activity. Here we report on three such properties. During ex vivo murine bone marrow erythroid development, compared directly to rHuEPO, peginesatide first enhanced KitposCD71pos progenitor cell expansion (including KitposCD71lowCD36posCD13pos erythromyelo- progenitors). Second, peginesatide exhibited a 1300 minute EPOR residence time vs. 77 minutes for rHuEPO. Third, the culture of EPO-dependent human UT7epo cells in peginesatide led to substantial EPOR up-modulation, as well as an apparent lessening of the processing of mature EPOR's. Furthermore, in studies that compared peginesatide vs. rHuEPO effects on the erythroid development of human bone marrow-derived CD34pos progenitors, the following differences were observed. At days 6–10 of culture, peginesatide gave rise to increased frequencies (up to 200% as compared to rHuEPO) of KitposCD71pos co-positive erythroid progenitors. At early stages (d2-d4), cell-surface EPOR levels also were elevated among progenitors expanded in peginesatide. When levels of CD13posCD36pos co-positive cells were analyzed, frequencies of these erythromyelo-progenitors also were heightened up to three-fold. Analyses of cultures at later time-points (eg, d10 of culture) indicated that peginesatide and rHuEPO supported the formation of GPAhigh erythroblasts at similarly high frequencies. In addition, cytospin analyses revealed morphological distinctions for multicellular proerythroblast complexes formed in peginesatide. Peginesatide's persistent erythropoietic activity allowing for once-monthly dosing therefore is likely to involve novel effects on erythromyelo-progenitor recruitment, and increased EPOR cell surface expression including apparent increases in relative levels of full-length EPOR forms. Disclosures: Green: Affymax, Inc.: Employment. Leu:Affymax, Inc.: Employment. Mortensen:Affymax, Inc.: Employment. Young:Affymax, Inc.: Prior employment Other. Schatz:Affymax, Inc.: Employment. Wojchowski:Affymax, Inc.: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Erythroid cell growth from normal and W/WV murine bone marrow on macrophage-coated membranes

Blood ◽  
1977 ◽  
Vol 50 (5) ◽  
pp. 857-866
Author(s):  
BJ Torok-Starb ◽  
NS Wolf ◽  
DR Boggs
Keyword(s):  
Bone Marrow ◽  
Cell Growth ◽  
Bone Marrow Cells ◽  
Erythroid Cell ◽  
Erythroid Progenitor ◽  
Murine Bone Marrow ◽  
Cellulose Acetate Membranes ◽  
Coated Membranes ◽  
Marrow Cells

Cellulose acetate membranes (CAM) placed in the peritoneal cavity of mice develop a macrophage layer capable of supporting in vivo hematopoietic colonies from intraperitoneally injected bone marrow cells. Modifications allowing for routine morphologic identification of colonies showed that both erythrocytic (E) and granulocytic (G) colonies occur with a consistent E:G ratio of 0.19 +/- 0.037. Stimulating recipients by bleeding or phenylhydrazine injection did not produce a significant change in the total number of colonies and a reduction in granulocytic colonies so that the E:G ratio significnatly increased. Hypertransfusion of donor animals had no effect on the number of erythroid colonies that grew on CAM of average recipients. The total colony-forming ability of bone marrow cells from genetically anemic W/WV mice was found not to differ from that of normal +/+ littermates; however, the E:G ratio of W/WV marrow in bled recipients was significantly lower (p less than 0.01) then that of +/+ marrow. These studies suggest that a CAM system supports an erythroid progenitor which is not affected by hypotransfusion of the donor animal, yet is dependent upon erythropoietin for colony formation, and that it is defective in the W/WV mouse.

ATG5 and ATG7 Fulfill Essential and Non-Redundant Roles in Human Hematopoietic Stem/Progenitor Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4316-4316
Author(s):  
Hendrik Folkerts ◽  
Maria Catalina Gomez Puerto ◽  
Albertus T.J. Wierenga ◽  
Koen Schepers ◽  
Jan Jacob Schuringa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Progenitor Cells ◽  
Target Genes ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Human Hscs

Abstract Macroautophagy is a catabolic process by which intracellular contents are delivered to lysosomes for degradation. ATG5 and ATG7 play an essential role in this process. Recent studies have shown that mouse hematopoietic stem cells (HSCs) lacking ATG7 were unable to survive in vivo, however, the role of macroautophagy in proliferation and survival of human HSCs has not yet been defined. Here, we demonstrate that autophagy is functional in human hematopoietic stem/progenitor cells. Robust accumulation of the autophagy markers LC3 and p62 were observed in cord blood (CB)-derived CD34+ cells treated with bafilomycin-A1 (BAF) or hydroxychloroquine (HCQ), as defined by Western blotting. When these cells were subsequently differentiated towards the myeloid or erythroid lineage, a decreased accumulation of LC3 was observed. In addition, CB CD34+CD38- cells showed enhanced accumulation of cyto-ID (a marker for autophagic vesicles) compared to CD34+CD38+ progenitor cells upon BAF or HCQ treatment. In line with these results, also more mature CB CD33+ and CD14+ myeloid cells or CD71+CD235+ erythroid cells showed reduced levels of cyto-ID accumulation upon BAF or HCQ treatment. These findings indicate that human hematopoietic stem and progenitor cells (HSPCs) have a higher basal autophagy flux compared to more differentiated cells. To study the functional consequences of autophagy in human HSCs and their progeny, ATG5 and ATG7 were downregulated in CB-derived CD34+ cells, using a lentiviral shRNA approach which resulted in 80% and 70% reduced expression, respectively. Downmodulation of ATG5 or ATG7 in CB CD34+ cells resulted in a significant reduction of erythroid progenitor frequencies, as assessed by colony forming cell (CFC) assays (shATG5 2.2 fold, p<0.05 or shATG7 1.4 fold p<0.05). Additionally, a strong reduction in expansion was observed when transduced cells were cultured under myeloid (shATG5 17.9 fold, p<0.05 or shATG7 12.3 fold, p<0.05) or erythroid permissive conditions (shATG5 6.7 fold, p<0.05 or shATG7 1.7 fold, p<0.05), whereby differentiation was not affected. The phenotype upon knockdown of ATG5 or ATG7 could not be reversed by culturing the cells on a MS5 stromal layer. In addition to progenitor cells, HSCs were also affected since long term culture-initiating cell (LTC-IC) assays in limiting dilution revealed a 3-fold reduction in stem cell frequency after ATG5 and ATG7 knockdown. The inhibitory effects of shATG5 and shATG7 in cultured CD34+ cells were at least in part due to a decline in the percentage of cells in S phase and (shATG5 1.4 fold, p<0.01 and shATG7 1.3 fold, p<0.01) and an increase of Annexin V positive cells. The changes in cell cycle and apoptosis coincided with a marked increase in expression of the cell cycle-dependent kinase inhibitor p21, an increase in p53 levels, and an increase in proapoptotic downstream target genes BAX, PUMA and PHLDA3. Additionally, ROS levels were increased after ATG5 and ATG7 knockdown. The increased apoptosis in shATG5 and shATG7 transduced cells might be triggered by elevated ROS levels. Taken together, our data demonstrate that autophagy is an important survival mechanism for human HSCs and their progeny. Disclosures No relevant conflicts of interest to declare.

scholarly journals Functional Requirements of a Samd14-Capping Protein Interaction in Stress Erythropoiesis

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 288-288
Author(s):  
Suhita Ray ◽  
Linda Chee ◽  
Nicholas T. Woods ◽  
Kyle J Hewitt
Keyword(s):  
Steady State ◽  
Hemolytic Anemia ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Interacting Proteins ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Capping Protein ◽  
Sam Domain ◽  
Stress Erythropoiesis

Abstract Stress erythropoiesis describes the process of accelerating red blood cell (RBC) production in anemia. Among a number of important mediators of stress erythropoiesis, paracrine signals - involving cooperation between SCF/c-Kit signaling and other signaling inputs - are required for the activation/function of stress erythroid progenitors. Whereas many critical factors required to drive erythropoiesis in normal physiological conditions have been described, whether distinct mechanisms control developmental, steady-state, and stress erythropoiesis in anemia is poorly understood. Our prior work revealed that the Sterile Alpha Motif (SAM) Domain 14 (Samd14) gene is transcriptionally upregulated in a model of acute hemolytic anemia induced by the RBC-lysing chemical phenylhydrazine. Samd14 is regulated by GATA binding transcription factors via an intronic enhancer (Samd14-Enh). In a mouse knockout of Samd14-Enh (Samd14-Enh -/-), we established that the Samd14-Enh is dispensable for steady-state erythropoiesis but is required for recovery from severe hemolytic anemia. Samd14 promotes c-Kit signaling in vivo and ex vivo, and the SAM domain of Samd14 facilitates c-Kit-mediated cellular signaling and stress progenitor activity. In addition, the Samd14 SAM domain is functionally distinct from closely related SAM domains, which demonstrates a unique role for this SAM domain in stress signaling and cell survival. In our working model, Samd14-Enh is part of an ensemble of anemia-responsive enhancers which promote stress erythroid progenitor activity. However, the mechanism underlying Samd14's role in stress erythropoiesis is unknown. To identify potential Samd14-interacting proteins that mediate its function, we performed immunoprecipitation-mass spectrometry on the Samd14 protein. We found that Samd14 interacted with α- and β heterodimers of the F-actin capping protein (CP) complex independent of the SAM domain. CP binds to actin during filament assembly/disassembly and plays a role in cell morphology, migration, and signaling. Deleting a 17 amino acid sequence near the N-terminus of Samd14 disrupted the Samd14-CP interaction. However, mutating the canonical RxR of the CP interaction (CPI) motif, which is required for CP-binding in other proteins, does not abrogate the Samd14-CP interaction. Moreover, replacing this sequence with the canonical CPI domain of CKIP-1 completely disrupts the interaction, indicating that other sequence features are required to maintain the Samd14-CP complex. Ex vivo knockdown of the β-subunit of CP (CPβ), which disrupts the integrity of the CP complex, decreased the percentage of early erythroid precursors (p&lt;0.0001) and decreased (3-fold) progenitor activity as measured by colony formation assays (similar to knockdown of Samd14). Taken together, these data indicate that Samd14 interacts with CP via a unique CP binding (CPB) domain, and that the CP complex coordinates erythroid differentiation in stress erythroid progenitors. To test the function of the Samd14-CP complex, we designed an ex vivo genetic complementation assay to express Samd14 lacking the CPB-domain (Samd14∆CPB) in stress erythroid progenitors isolated from anemic Samd14-Enh -/- mice. Phospho-AKT (Ser473) and phospho-ERK (Thr202/Tyr204) levels in Samd14∆CPB were, respectively, 2.2 fold (p=0.007) and ~7 fold (n=3) lower than wild type Samd14 expressing cells, 5 min post SCF stimulation. Relative to Samd14, Samd14∆CPB expression reduced burst forming unit-erythroid (BFU-E) (2.0 fold) and colony forming unit-erythroid (CFU-E) (1.5 fold). These results revealed that the Samd14-CP interaction is a determinant of BFU-E and CFU-E progenitor cell levels and function. Remarkably, as the requirement of the CPB domain in BFU-E and CFU-E progenitors is distinct from the Samd14-SAM domain (which promotes BFU-E but not CFU-E), the function of Samd14 in these two cell types may differ. Ongoing studies will examine whether the function of Samd14 extends beyond SCF/c-Kit signaling and establish cell type-dependent functions of Samd14 and Samd14-interacting proteins. Given the critical importance of c-Kit signaling in hematopoiesis, the role of Samd14 in mediating pathway activation, and our discovery implicating the capping protein complex in erythropoiesis, it is worth considering the pathological implications of this mechanism in acute/chronic anemia and leukemia. Disclosures No relevant conflicts of interest to declare.

The Acute Promyelocytic Leukemia-Oncoprotein PML-Raralpha Blocks Senescence and Disrupts The Atrx/Daxx Chromatin Remodeling Complex To Promote Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1267-1267
Author(s):  
Katharina Korf ◽  
Harald Wodrich ◽  
Alexander Haschke ◽  
Ron M. Evans ◽  
Thomas M. Sternsdorf
Keyword(s):  
Bone Marrow ◽  
Acute Promyelocytic Leukemia ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Cell Cycle Regulators ◽  
Murine Bone Marrow ◽  
Pml Nuclear Bodies

Abstract Although Acute Promyelocytic Leukemia (APL) has become a curable disease due to in-depth understanding of the underlying molecular processes, its investigation has provided unique and valuable insights into the processes involved in leukemogenesis. Therefore we use it as a model disease. 99% of APL-patients express a PML-RAR fusion protein. While involvement of RAR has proven indispensable for oncogenicity, the role of the PML domain is far less clear. In our previous study (Sternsdorf et al., Cancer Cell, 2006) we found that substitution of PML with heterologous self-interaction domains suffices to induce leukemias, but drastically decreases oncogenic potency of the resulting fusion proteins. In this study, we have chosen the inverse strategy: we have modified the PML domain to create a more active artificial model oncoprotein by adapting PR to its biological environment: As the typical model organism for APL studies is the mouse, we have replaced the human PML domain with the murine PML domain. This oncoprotein (mPR) creates APL-type leukemias in mice with higher penetrance and shorter latency than its human counterpart, hPR. We have used this system to study immediate early effects of expression of the model oncoprotein. While proliferating murine bone marrow cells go into senescence ex vivo, expression of mPR prevents this and robustly immortalizes murine bone marrow from every mouse strain tested so far. Senescence-associated upregulation of the cell-cycle regulators p21 and p19 was efficiently blocked by mPR expression. In mouse cells, mPR exhibits higher potency in disrupting the PML-associated Daxx/ATRX complex than hPR. Knockdown of ATRX, but not Daxx ameliorated ATRA-induced growth suppression and p21 upregulation in the human APL model cell line NB4. These data suggest, that PML-RAR promotes leukemogenesis by disrupting the Daxx/ATRX complex, which assembles at PML nuclear bodies during the onset of senescence. Disclosures: No relevant conflicts of interest to declare.

Early Erythroid Development Is Enhanced with Hypoxia and Terminal Maturation with Normoxia in a 3D Ex Vivo Physiologic Eythropoiesis Model

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2453-2453
Author(s):  
Susana Brito dos Santos ◽  
Mark C. Allenby ◽  
Athanasios Mantalaris ◽  
Nicki Panoskaltsis
Keyword(s):  
Mononuclear Cells ◽  
Cytokine Production ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Erythroid Progenitors ◽  
Variable Intensity ◽  
Erythroid Progenitor ◽  
Tnf Α ◽  
The Impact

Abstract Reproduction of dynamic physiologic erythropoiesis in vitro requires a three-dimensional (3D) architecture, erythroblast-macrophage interactions and cytokines such as erythropoietin (EPO). The role of oxygen concentration gradients in this process is unclear. We have created a 3D bone marrow (BM) biomimicry using collagen-coated polyurethane scaffolds (5mm3) to expand cord blood mononuclear cells (CBMNCs) in a cytokine-free environment for 28 days (D). Addition of EPO to this system induces mature erythropoiesis. We hypothesised that physiologic concentrations of cytokines - stem cell factor (SCF) / EPO - and a hypoxia (H)/normoxia (N) schedule to mimic BM oxygen gradients would enhance erythropoiesis. CBMNCs were seeded (4x106 cells/scaffold) in 3D serum-free cultures supplemented with 10ng/mL SCF (D0-D28), and 100mU/mL EPO (D7-D28), with medium exchange every 3D. Three conditions were compared: N (20%), H (5%) and 2-step oxygenation HN (H D0-D7 and N thereafter). Erythroid maturation was monitored weekly by flow cytometry (CD45/CD71/CD235a) both in situ (i.e., in scaffolds) and in supernatant (S/N) cells. D0-7 H was more efficient in early induction of CD235a in the absence of exogenous EPO (H 13% vs N 8% CD45loCD71+CD235alo cells, p<0.05). This maturation profile was also observed in D10 S/N cells, in which CD45loCD71+CD235a+ cells were proportionately more in H (30%) and HN (27%) than in N (16%, p<0.05). By D14, N and HN stimulated the appearance of CD45-CD71+CD235a+ cells, whereas H maintained the CD45loCD71+CD235a-/lo phenotype. By D21, a CD45-CD71+CD235a+ mature population was clearly distinguished in all conditions, most notably in N (16%) and HN (21%) vs H (9%). At D28, more mature CD45-CD71loCD235a+ cells were observed in normoxia conditions, N 3% and HN 4%, vs H 0.3%. A renewed population of erythroid progenitors was also evident at this time (H 62%, N 51% and HN 46% CD45loCD71lo/+CD235a- cells). In order to assess the impact of H and N on erythroid gene transcription, we evaluated erythroid signatures by qRT-PCR. GATA-1 expression was detected from D7, highest for H at D14 (p<0.05), and decreased thereafter. GATA-2 expression was up-regulated only at D28, in particular in N (p<0.05), and correlated with emerging erythroid progenitors identified at this stage. At D14, EPOR expression was maximal, especially in HN (p<0.05), simultaneous with high pSTAT5 levels, suggesting activation of EPOR signalling. Also at D14, H upregulated γ-globin (p<0.05). By Western Blot, only H and HN still produced γ-globin whereas β-globin expression was clearly detected in all conditions by D28. In situ production of cytokines was evaluated by cytometric bead array in the exhausted media. IL-6, G-CSF, GM-CSF, IL-1, TNF-α and IL-17 were detected at higher concentrations during the first 7 days, declining to undetectable thereafter. IL-21 was not detected at any point. IL-3 was detected from D13, with highest expression in H (p<0.05, D22). VEGF was also expressed after D7, highest in H (p<0.05, D16 & D19), concurrent with HIF-1α up-regulation observed at D7 and D14. TNF-α was produced with variable intensity from D4. These data suggested that D7-D14 was a crucial period for culture dynamics, in particular for H and HN, with up-regulation of erythroid transcription factors, EPOR signalling, and endogenous cytokine production. BFU-E and CFU-E also dominated the first 14 days of culture. Scanning electron microscopy at D17 and D25 revealed niche-like structures in situ, which expressed STRO-1, osteopontin and vimentin at D19 by confocal immunofluorescent microscopy, indicative of an endogenous stromal cell microenvironment. CD68+ cells were also detected at D19 in proximity to CD71+ cells suggesting formation of erythroblastic islands. In this 3D ex vivo biomimicry using near-physiologic cytokine and oxygen conditions, H induced initial erythroid commitment and established an early erythroid progenitor population. N was required at later maturational stages and enhanced the γ-globin to β-globin switch. We identified D7-D14 as a crucial timeframe in this system wherein endogenous cytokine production as well as up-regulation of GATA-1, EPOR and HIF-1α was observed. We propose that a combined HN schedule in this 3D BM biomimicy may enable a more robust and physiologic culture platform to study normal and abnormal erythroid differentiation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Lentiviral shRNA silencing of murine bone marrow cell CCR2 leads to persistent knockdown of CCR2 function in vivo

Blood ◽  
2005 ◽  
Vol 106 (4) ◽  
pp. 1147-1153 ◽  
Author(s):  
Ilze Bot ◽  
Jian Guo ◽  
Miranda Van Eck ◽  
Peter J. Van Santbrink ◽  
Pieter H. E. Groot ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Function ◽  
Chemokine Receptors ◽  
Ex Vivo ◽  
Marrow Cell ◽  
Major Barrier ◽  
Murine Bone Marrow ◽  
Ccr2 Expression

AbstractA major barrier in hematopoietic gene function studies is posed by the laborious and time-consuming generation of knockout mice with an appropriate genetic background. Here we present a novel lentivirus-based strategy for the in situ generation of hematopoietic knockdowns. A short hairpin RNA (shRNA) was designed targeting murine CC-chemokine receptor 2 (CCR2), which was able to specifically blunt CCR2 expression at the mRNA, protein, and functional levels in vitro. Reconstitution of irradiated recipient mice with autologous bone marrow that had been ex vivo transduced with shRNA lentivirus led to persistent down-regulation of CCR2 expression, which translated into a 70% reduction in CCR2-dependent recruitment of macrophages to an inflamed peritoneal cavity without noticeable side effects on related chemokine receptors or general inflammation status. These findings clearly demonstrate the potential of shRNA lentivirus–infected bone marrow transplantation as a rapid and effective method to generate hematopoietic knockdowns for leukocyte gene function studies.

scholarly journals Complete Spatial Mapping of Steady-State and Stress Erythropoiesis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 7-7
Author(s):  
Qingqing Wu ◽  
Jizhou Zhang ◽  
Courtney Johnson ◽  
Anastasiya Slaughter ◽  
Margot Lindsay May ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Single Cells ◽  
Differentially Expressed ◽  
Published Data ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Clonal Architecture ◽  
Stress Erythropoiesis

The anatomy of differentiation in the bone marrow (BM) is poorly understood due to lack of markers to image stepwise HSPC differentiation. We analyzed 250+ cell surface molecules in all hematopoietic progenitors and identified 56 differentially expressed markers in at least one HSPC that can be "mixed and matched" to prospectively image any HSPC of interest in the bone marrow. We used this data to develop a pipeline to map stepwise erythropoiesis in vivo. We found that all erythroid progenitors can be defined as Ly6C-CD27-ESAM-CD117+ cells and then Pre-MegE (earliest erythroid progenitor Cell Stem Cell. 2007 1(4):428-42) are CD150+CD71-. These give rise to CD71+CD150+ Pre-CFU-E that differentiate into CD71+CD150- CFU-E that then generate early erythroblasts. All BFU-E activity was restricted to Pre-MegE and Pre- CFU-E (70 and 30% of all BFU-E) whereas all CFU-E colonies were spread between Pre-MegE (44%), pre-CFU-E (10%) and CFU-E (46%). We also confirmed previously published data showing that CD71 and Ter119 can be used to image stepwise terminal erythropoiesis; CD71+Ter119dim early erythroblasts, CD71+Ter119bright late erythroblasts, CD71dimTer119bright reticulocytes and CD71-Ter119bright erythrocytes. Importantly, all populations were detected at identical frequencies using FACS or confocal imaging indicating that our imaging strategy detects all erythroid cells (Pre-CFU-E: 0.022 vs 0.027 %; CFUE: 0.32 vs 0.30%; Early-Ery: 0.62 vs 0.66%; Late-Ery: 32.05 vs 32.12%; Reticulocyte: 5.98 vs. 3.36%; Erythrocytes: 12.49 vs. 13.47%). We mapped the 3D location of every erythroid lineage cell in mouse sternum and interrogated the spatial relationships between the different maturation steps and with candidate niches. We compared the interactions found in vivo with those found in random simulations. Specifically, we used CD45 and Ter119 to obtain the spatial coordinates of every hematopoietic cell. Then we randomly placed each type of erythroid lineage cell at identical frequencies as those found in vivo to generate random simulations. We found erythroid progenitors show no specific association with HSC, indicating that Pre-Meg-E or more primitive progenitors leave the HSC niche after differentiation. Both Pre-Meg-E and Pre-CFU-E are found as single cells through the central BM space and do not specifically associate with other progenitors, or components of the microenvironment. In contrast almost all CFU-E locate to strings (28 strings per sternum) containing 8 CFU-E that are selectively recruited to sinusoids (mean CFU-E to sinusoid distance=2.2µm). As soon as CFU-E detach from sinusoids they downregulate CD117 and upregulate CD71 giving rise to a cluster of early erythroblasts that buds from the vessel. These progressively upregulate Ter119 to generate large clusters of late erythroblasts that in turn differentiate into clusters of reticulocytes and erythrocytes. To examine the clonal architecture of erythropoiesis we used Ubc-creERT2:confetti mice where a tamoxifen pulse leads to irreversible expression of GFP, CFP, YFP or RFP. Four weeks later we found that the CFU-E strings are oligoclonal with each clone contributing 2-6 CFU-E to the string. The budding erythroblasts clusters are similarly organized. These indicate that different CFU-E are serially recruited to the same sinusoidal spot where they self-renew 1-2 times and then undergo terminal differentiation. We then tracked how this architecture changed in response to stress (hemorrhage). Two days after bleeding we found that Pre-Meg-E and Pre-CFU-E numbers and locations were unaltered. The number of CFU-E strings remained constant (30 CFUE strings/sternum) but all strings contained more CFU-E (2-fold) suggesting increased self-renewal. Unexpectedly, fate mapping showed that the size of CFU-E clones did not increase when compared to steady-state. These results indicate that all CFU-E expand in respond to stress and that this is mediated via increased recruitment and differentiation of upstream progenitors. In summary we have found 56 differentially expressed markers that can be combined to detect most HSPC; validated a 5-color stain to image and fate map all steps of red blood cell maturation in situ; demonstrated that terminal erythropoiesis emerges from strings of sinusoidal CFU-E, and revealed the clonal architecture of normal and stress erythropoiesis. Disclosures No relevant conflicts of interest to declare.

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