The residual megakaryocyte and platelet production in c-Mpl–deficient mice is not dependent on the actions of interleukin-6, interleukin-11, or leukemia inhibitory factor

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 528-534 ◽  
Author(s):  
Timothy Gainsford ◽  
Harshal Nandurkar ◽  
Donald Metcalf ◽  
Lorraine Robb ◽  
C. Glenn Begley ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Leukemia Inhibitory Factor ◽  
Double Mutant ◽  
Essential Role ◽  
Physiological Role ◽  
Mutant Mice ◽  
Platelet Production ◽  
Interleukin 11 ◽  
Deficient Mice

Mice lacking thrombopoietin (TPO) or its receptor c-Mpl are severely thrombocytopenic, consistent with a dominant physiological role for this cytokine in megakaryocytopoiesis. However, these mice remain healthy and show no signs of spontaneous hemorrhage, implying that TPO-independent mechanisms for platelet production exist and are sufficient for hemostasis. To investigate the roles of cytokines that act through the gp130 signaling chain in the residual platelet production of mpl-/- mice, mpl-/-IL-6-/-, mpl-/-LIF-/-, andmpl-/-IL-11R-/-double-mutant mice were generated. In each of these compound mutants, the number of circulating platelets was no lower than that observed in mice lacking only the c-mpl gene. Moreover, the deficits in the numbers of megakaryocytes and megakaryocyte progenitor cells in the bone marrow and spleen were no further exacerbated inmpl-/-IL-6-/-,mpl-/-LIF-/-, ormpl-/-IL-11R-/-double-mutant mice compared with those in Mpl-deficient animals. In single IL-6-/-, LIF-/-, andIL-11R-/- mutant mice, platelet production was normal. These data establish that, as single regulators, IL-6, IL-11, and LIF have no essential role in normal steady-state megakaryocytopoiesis, and are not required for the residual megakaryocyte and platelet production seen in thec-mpl-/- mouse.

scholarly journals Cytokine Production and Function in c-mpl–Deficient Mice: No Physiologic Role for Interleukin-3 in Residual Megakaryocyte and Platelet Production

Blood ◽  
1998 ◽  
Vol 91 (8) ◽  
pp. 2745-2752 ◽  
Author(s):  
Timothy Gainsford ◽  
Andrew W. Roberts ◽  
Shinya Kimura ◽  
Donald Metcalf ◽  
Glenn Dranoff ◽  
...  
Keyword(s):  
Leukemia Inhibitory Factor ◽  
Double Mutant ◽  
Potential Role ◽  
Cytokine Production ◽  
Genetic Defects ◽  
Platelet Production ◽  
And Function ◽  
Physiologic Role ◽  
Deficient Mice

Mice lacking thrombopoietin (TPO), or its receptor c-Mpl, display defective megakaryocyte and platelet development and deficiencies in progenitor cells of multiple hematopoietic lineages. The contribution of alternative cytokines to thrombopoiesis in the absence of TPO signalling was examined in mpl−/− mice. Analysis of serum and organ-conditioned media showed no evidence of a compensatory overproduction of megakaryocytopoietic cytokines. However, consistent with a potential role in vivo, when injected intompl−/− mice, interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) retained the capacity to elevate megakaryocytes and their progenitors in hematopoietic tissues and increase circulating platelet numbers. However, double mutant mice bred to carry genetic defects both in c-Mpl and IL-3 or the alpha chain of the IL-3 receptor, displayed no greater deficiencies in megakaryocytes or platelets than mpl-deficient animals, suggesting absence of a physiologic role for IL-3 in the residual megakaryocytopoiesis and platelet production in these mice.

Enhanced Erythroblast Mobilization in Nix-Deficient Mice Confers Resistance to Phenylhydrazine-Induced Anemia Despite Accelerated Erythrocyte Turnover.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3659-3659
Author(s):  
Abhinav Diwan ◽  
Andrew G. Koesters ◽  
Amy M. Odley ◽  
Theodosia A. Kalfa ◽  
Gerald W. Dorn
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Half Life ◽  
Hematopoietic Cells ◽  
Dynamic Regulation ◽  
Genetic Ablation ◽  
Deficient Mice ◽  
Erythrocyte Fragility

Abstract Steady-state and dynamic regulation of erythrocyte production occurs by altering the balance of cell-survival versus apoptosis signaling in maturing erythroblasts. Previously, the pro-apoptotic factor Nix was identified as a critical death signal in normal erythropoietic homeostasis, acting in opposition to erythroblast-survival signaling by erythropoietin and Bcl-xl. However, the role of Nix in stress-erythropoiesis is not known. Here, by comparing the consequences of erythropoietin administration, acute phenylhydrazine-induced anemia, and aging in wild-type and Nix-deficient mice, we show that complete absence of Nix, or its genetic ablation specifically in hematopoietic cells, mimics the effects of erythropoietin (Epo). Both Nix ablation and Epo treatment increase early erythroblasts in spleen and bone marrow and increase the number of circulating reticulocytes, while maintaining a pool of mature erythroblasts as an “erythropoietic reserve”. As compared with WT, Nix null mice develop polycythemia more rapidly after Epo treatment, consistent with enhanced sensitivity to erythropoietin observed in vitro. After phenylhydrazine administration, anemia in Nix-deficient mice is less severe and recovers more rapidly than in WT mice, despite lower endogenous Epo levels. Anemic stress depletes mature erythroblasts in both WT and Nix null mice, but Nix null mice with basal erythroblastosis are resistant to anemic stress. These findings show that Nix null mice have greatly expanded erythroblast reserve and respond normally to Epo- and anemia-stimulated induction of erythropoiesis. However, the hematocrits of young adult Nix null mice are not elevated, and these mice paradoxically develop anemia as they age with decreased hemoglobin content (10g/dl) and hematocrit (36%; at 80±3 weeks of age) compared to WT mice (13g/dl and 46%; 82±5 weeks of age), inspite of persistent erythoblastosis observed in the bone marrow and spleen. Nix null erythrocytes, which are macrocytic and exhibit membrane abnormalities typically seen in immature cells or with accelerated erythropoiesis, demonstrate shorter life span with a half life of 5.2±0.6 days in the peripheral circulation by in vivo biotin labeling (as compared with a half life of 11.7±0.9 days in WT), and increased osmotic fragility as compared with normal erythrocytes. This suggests that production and release of large numbers of reticulocytes in Nix null mice can decrease erythrocyte survival. To rule out a non-hematopoietic consequence of Nix ablation that contributes to or causes increased erythrocyte fragility and in vivo consumption, such as primary hypersplenism, we undertook Tie2-Cre mediated conditional Nix gene ablation. Nixfl/fl + Tie2-Cre mice (hematopoietic-cell specific Nix null) develop erythroblastosis with splenomegaly, reticulocytosis, absence of polycythemia and increased erythrocyte fragility; suggesting that erythroblastosis and accelerated erythrocyte turnover are a primary consequence of Nix ablation in hematopoietic cells. Hence, dis-inhibition of erythropoietin-mediated erythroblast survival pathways by Nix ablation enhances steady-state and stress-mediated erythropoiesis.

PU.1 and p53 Double Mutant Mice Develop Aggressive AML with Dysplastic Features

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 769-769
Author(s):  
Petra Vlckova ◽  
Libor Stanek ◽  
Pavel Burda ◽  
Karin Vargova ◽  
Filipp Savvulidi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Progenitor Cells ◽  
Double Mutant ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Regulatory Element ◽  
Tumour Suppressor ◽  
Mutant Mice ◽  
Stem And Progenitor Cells

Abstract Abstract 769 Introduction: Downregulation of tumour suppressor transcription factor PU.1 in haematopoietic stem and progenitor cells represents primary underlying mechanism for the development of acute myeloid leukaemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. Human AML often display differences in aggressiveness that are associated with mutations of a well known tumour suppressor p53. We produced murine model carrying mutations of p53 and URE that develops highly aggressive AML and focused on molecular mechanisms that are responsible for AML aggressiveness. Mouse models: PU.1ure/ure (Rosenbauer F, et al. 2004) and p53−/− (Jacks T, et al. 1994) mice were used. Conditional deletion of the URE leads to downregulation of PU.1 and is marked by clonal accumulation of myeloid c-Kit+Mac-1low Gr-1low blast cells within bone marrow, spleen, and peripheral blood mirrored by lower numbers of lymphoid and erythroid cells. AML development in PU.1ure/ure mice involves a preleukaemic phase (at 2–3 months) marked by proliferation of myeloid c-Kit+Gr-1+ cells and splenomegaly. Interestingly, p53−/−mice do not develop AML, instead loss of p53 predisposes mice to solid tumours, mostly lymphomas, by 6 months of age. Results: Deletion of TP53 in the PU.1ure/ure mice (PU.1ure/ure p53−/−) results in more aggressive AML with significantly shortened overall survival, prominent hepatosplenomegaly and cachexia (wasting syndrome). Mild differences in cell surface phenotype of bone marrow derived cells were observed between PU.1ure/ure and PU.1ure/ure p53−/− mice by flow cytometry (these included: blasts expansion and lymphopenia). Next, the PU.1 expression was determined in all genotypes at progenitor and stem cell levels. PU.1 mRNA level in more aggressive PU.1ure/ure p53−/− murine AML is decreased in the entire c-Kit+tumour cell population compared to AML in PU.1ure/ure mice including haematopoietic stem and progenitor cells (HSPCs). Correspondingly to RNA level, in the PU.1ure/ure progenitors the PU.1 protein was decreased compared to p53−/− progenitors and is yet further reduced in the PU.1ure/ure p53−/− c-Kit+ Mac1+progenitors. p53−/− progenitors express similar level of PU.1 as wild type progenitors indicating that despite p53 can bind DNA as a transcription factor, it does not regulate PU.1 level directly. In addition to URE deletion we searched for other mechanisms that control PU.1 levels and found that PU.1-inhibiting microRNA miR-155 gene display altered chromatin structure and expression of both pri-miR-155 as well as its spliced mature form in the AML of PU.1ure/ure and (to higher extent in) PU.1ure/ure p53−/− murine progenitors. Upregulation of miR-155 coincides with upregulation of the Mir155hg activators: Myc and Myb. Finally, upon inhibition of either Myb or miR-155 in vitro the AML progenitors restore PU.1 levels and lose leukaemic cell growth. Conclusion: In summary, PU.1 and p53 double mutant mice develop aggressive AML with dysplastic features. Defective control of PU.1 levels in PU.1ure/ure and PU.1ure/ure p53−/−AML involves miR-155. Lastly, restored PU.1 level and cell differentiation capacity are achieved by inhibiting either Myb or miR-155 in the PU.1ure/ure p53−/− progenitors. (Grant support: P305/12/1033, UNCE 204021, PRVOUK-P24/LF1/3, SVV-2012-264507, P301/12/P380. MK was sponsored by GAUK 251070 45410, 251135 82210) Disclosures: No relevant conflicts of interest to declare.

Disturbed Endothelial Blood-Bone Marrow-Barrier In Nox4 Deficient Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1169-1169
Author(s):  
Maren Weisser ◽  
Kerstin B. Kaufmann ◽  
Tomer Itkin ◽  
Linping Chen-Wichmann ◽  
Tsvee Lapidot ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Stem Cell ◽  
Steady State ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Knock Out ◽  
Deficient Mice ◽  
Permeability State

Abstract Reactive oxygen species (ROS) have been implicated in the regulation of stemness of hematopoietic stem cells (HSC). HSC with long-term repopulating capabilities are characterized by low ROS levels, whereas increased ROS levels correlate with lineage specification and differentiation. Several tightly regulated sources of ROS production are well known among which are the NADPH oxidases (Nox). HSC are known to express Nox1, Nox2 and Nox4, however, their role in maintenance of stem cell potential or in the activation of differentiation programs are poorly understood. While Nox2 is activated in response to various extrinsic and intrinsic stimuli, mainly during infection and inflammation, Nox4 is constitutively active and is considered to be responsible for steady-state ROS production. Consequently, Nox4 deficiency might lower ROS levels at steady-state hematopoiesis and thereby could have an impact on HSC physiology. In this work we studied HSC homeostasis in Nox4 knock-out mice. Analysis of the hematopoietic stem and progenitor cell (HSPC) pool in the bone marrow (BM) revealed no significant differences in the levels of Lineage marker negative (Lin-) Sca-1+ ckit+ (LSK) and LSK-SLAM (LSK CD150+ CD48-) cells in Nox4 deficient mice compared to wild type (WT) C57BL/6J mice. HSPC frequency upon primary and secondary BM transplantation was comparable between Nox4 deficient and WT mice. In addition, the frequency of colony forming cells in the BM under steady-state conditions did not differ between both mouse groups. However, Nox4 deficient mice possess more functional HSCs as observed in in vivo competitive repopulating unit (CRU) assays. Lin- cells derived from Nox4 knock out (KO) mice showed an increased CRU frequency and superior multilineage engraftment upon secondary transplantation. Surprisingly, ROS levels in different HSPC subsets of NOX4 KO mice were comparable to WT cells, implying that the absence of Nox4 in HSCs does not have a major intrinsic impact on HSC physiology via ROS. Therefore, the increased levels of functional HSCs observed in our studies may suggest a contribution of the BM microenvironment to steady-state hematopoiesis in the BM of Nox4 KO animals. Recent observations suggest a regulation of the BM stem cell pool by BM endothelial cells, in particular by the permeability state of the blood-bone marrow-barrier (Itkin T et al., ASH Annual Meeting Abstracts, 2012). Endothelial cells interact with HSCs predominantly via paracrine effects and control stem cell retention, egress and homing as well as stem cell activation. As Nox4 is highly expressed in endothelial cells and is involved in angiogenesis, we reasoned that the absence of NOX4 could affect HSC homeostasis through altered BM endothelium properties and barrier permeability state. Indeed, in preliminary assays we found reduced short-term homing of BM mononuclear cells into the BM of Nox4 deficient mice as compared to wild type hosts. Furthermore, in vivo administration of Evans Blue dye revealed reduced dye penetration into Nox4-/- BM compared to wild type mice upon intravenous injection. Taken together, these data indicate a reduced endothelial permeability in Nox4 KO mice. Ongoing experiments aim at further characterization of the Nox4-/- phenotype in BM sinusoidal and arteriolar endothelial cells, the impact of Nox4 deletion on BM hematopoietic and mesenchymal stem cells, and in deciphering the role of Nox4 in the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals ROCK1 Functions As a Critical Regulator of Stress Erythropoiesis and Survival by Regulating p53

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 916-916
Author(s):  
Sasidhar Vemula ◽  
Jianjian Shi ◽  
Raghuveer Mali ◽  
Peilin Ma ◽  
Philip Hanneman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Steady State ◽  
Erythroid Cell ◽  
Cell Phenotype ◽  
Double Positive ◽  
Stress Erythropoiesis ◽  
Significant Difference ◽  
Rho Kinases ◽  
Deficient Mice

Abstract Abstract 916 Erythropoiesis is a dynamic, multistep process in which hematopoietic stem cells differentiate toward a progressively committed erythroid lineage through intermediate progenitors. Under normal physiologic condition, erythropoiesis takes place primarily in the bone marrow of humans and mice. Efficient stress erythropoiesis is crucial to survival and recovery from various pathophysiologic conditions including blood loss, anemia, and therapeutic procedures used in the treatment of hematological malignancies such as chemotherapy and stem cell transplantation. Impaired stress erythropoiesis can be fatal under these conditions. In order to develop improved strategies to stimulate stress erythropoiesis in patients with such conditions, it is critical to identify the molecular mechanisms that regulate this process. While many of the downstream signaling molecules and pathways have been elucidated in steady state erythroid cell development, the major regulators under stress conditions remain to be defined. The small families of Rho GTPases and their downstream effectors, Rho kinases have been implicated in regulating various cellular functions including actin cytoskeleton organization, cell adhesion, and cell motility in non-hematopoietic cells and inflammatory cells. Rho kinases (ROCK1 and ROCK2) belong to a family of serine/threonine kinases, and their physiologic role in erythropoiesis is not known. Utilizing mice deficient in the expression of ROCK1, we demonstrate no significant difference with respect to erythroid parameters in peripheral blood under steady-state conditions (n=19 mice for each genotype). The frequency of Ter119+, CD71+ or double-positive erythroid cells in the bone marrow and spleen of ROCK1-deficient mice was comparable to wild type (WT) controls at basal levels (n=7). In response to myelotoxic stress, ROCK1 deficient Ter119 positive cells demonstrated enhanced survival and recovery compared to WT controls (n=3–6, *p< 0.05). Further, using a phenylhydrazine (PHZ)-induced murine model of hemolytic anemia, we demonstrate that ROCK1-deficient mice exhibit increased red blood cells and enhanced hematocrits relative to WT mice (n=6, *p< 0.05). In addition, ROCK1−/− mice support efficient erythro-splenomegaly, and exhibit a threefold increase in splenic weight after PHZ injection compared to WT controls (n=6, *p< 0.05). Flow cytometric analysis revealed increased frequency of Ter119/CD71 double positive erythroid progenitor pool in ROCK1−/− spleens after PHZ treatment compared to WT (n=6, *p< 0.05). Furthermore, histopathological analysis of WT and ROCK1−/− spleens following PHZ treatment revealed a defined population of white pulp and red pulp in control spleens but enhanced extramedullary erythropoiesis in ROCK1−/− spleens (n=3, *p< 0.05). In vitro colony forming assays showed that ROCK1-deficient splenocytes generated more erythroid colony forming units (CFU-Es) and eventually generated more erythroid-burst forming units (BFU-Es) compared to WT splenocytes in response to different concentrations of EPO and combination of EPO and SCF (n=3, *p< 0.05). Deficiency of ROCK1 also resulted in enhanced survival of mice treated with PHZ compared to controls (n=17 mice per group, *p< 0.05). The enhanced survival of ROCK1-deficient mice in response to PHZ was associated with reduced reactive oxygen species (ROS) levels compared to WT (n=6 mice per group, *p< 0.05). Bone marrow transplantation studies revealed that enhanced stress erythropoiesis in ROCK1-deficient mice is stem cell autonomous (n=6 mice per genotype, *p< 0.05). Remarkably, the red cell phenotype observed in ROCK1−/− mice is similar to that reported in mice deficient in p53. We show that ROCK1 binds p53 directly and regulates its stability and expression (n=3). In absence of ROCK1, p53 phosphorylation and expression is reduced in ROCK1−/− erythroblasts (n=3). Our findings reveal that ROCK1 functions as a physiologic regulator of p53 under conditions of erythroid stress. These findings are expected to offer new perspectives on stress erythropoiesis and may provide a potential therapeutic target in human disease characterized by anemia. Disclosures: No relevant conflicts of interest to declare.

Knock-in of a FLT3 Internal Tandem Duplication Mutation Cooperates with a NUP98-HOXD13 Fusion to Generate Acute Leukemia in a Mouse Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2961-2961
Author(s):  
Sarah M Greenblatt ◽  
Li Li ◽  
Christopher Slape ◽  
David Huso ◽  
Peter D. Aplan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Mouse Model ◽  
Acute Leukemia ◽  
Double Mutant ◽  
Tandem Duplication ◽  
Mutant Mice ◽  
Leukemic Cells ◽  
Internal Tandem Duplication ◽  
Cell Counts

Abstract Abstract 2961 Poster Board II-937 Constitutive activation of FMS-like tyrosine kinase 3 (FLT3) by internal tandem duplication (ITD) is one of the most common molecular alterations in acute myeloid leukemia (AML), and provides a proliferative and survival advantage to leukemic cells. We have previously generated a knock-in mouse in which an 18-bp ITD mutation, isolated from a patient with AML, was inserted into the juxtamembrane domain of the murine FLT3 gene. Heterozygous FLT3WT/ITD mice develop a myeloproliferative disease, which progresses to mortality within 6 to 20 months. However, no sign of acute leukemia is observed over the lifetime of these mice, indicating that additional cooperating genetic events are required for leukemic progression. FLT3 activating mutations have been seen in MDS in about 5% of cases and an additional 5% of patients with MDS acquire FLT3 mutations as they progress to AML. One model of MDS has been developed by transgenic expression of the NUP98-HOXD13 (NHD13) fusion under the vav promoter. Mice expressing the NUP98-HOXD13 (NHD13) transgene develop a highly penetrant myelodysplastic syndrome (MDS) with about 50% eventually progressing to acute leukemia by 14 months. We wanted to generate a mouse model to see if FLT3/ITD mutations would cooperate with the NHD13 fusion to progress to overt leukemia. Double mutant mice (FLT3/ITD/NHD13) were generated by crossing heterozygous FLT3WT/ITD mice with mice expressing the NHD13 transgene. Strikingly, FLT3/ITD/NHD13 offspring (n=40) developed an acute leukemia with 100% penetrance and a median survival of 97 days. In contrast, NHD13 (n=20) and FLT3WT/ITD (n=20) littermates had median survivals of 385 and 410 days, respectively (Figure 1). Differential cell counts at the time of sacrifice showed elevated peripheral white blood cell counts in the double mutant mice (161.0±50.6 k/μl) compared to three-month-old wildtype (12.5 ±5.0 k/μl), NHD13(3.2 ±1.0 k/μl), or FLT3WT/ITD (7.9 ±3.5 k/μl) littermates. Organ sectioning and histological staining of double mutant mice showed leukemic infiltration of the spleen, liver, and brain. FLT3/ITD/NHD13 offspring developed a heterogeneous group of acute leukemias, with 46% developing ALL, 39% biphenotypic leukemia, and 15% AML as diagnosed by flow cytometry of the bone marrow and spleen. Transplantation experiments showed that the leukemia was able to engraft in lethally irradiated recipients, with disease occuring within 30 days post-transplantation. The disease was transferrable with as few as 1000 cells, and this ability was restricted to the B220+Mac-1−Gr-1- population. In order to identify the changes in gene expression responsible for leukemic transformation, RNA was isolated from the total bone marrow of young FLT3/ITD/NHD13, wildtype, NHD13, and FLT3WT/ITD littermates prior to the development of discernable disease and probed for over 43,000 coding and non-coding mouse sequences by Agilent 44K array. The rapid onset of acute leukemia in this model indicates the collaboration between hox gene dysregulation by a chromosomal translocation and altered signaling through the FLT3 receptor. In addition, since resistance to FLT3 inhibitors alone remains an important clinical issue, gene expression profiling of leukemic cells may help identify new molecular targets in collaborative signaling pathways. Disclosures: No relevant conflicts of interest to declare.

Loss of Foxo3 Reduces Erythroblast Apoptosis and Enhances RBC Production in Beta-Thalassemic Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 756-756 ◽  
Author(s):  
Raymond Liang ◽  
Genís Campreciós ◽  
Carolina L. Bigarella ◽  
Saghi Ghaffari
Keyword(s):  
Oxidative Stress ◽  
Bone Marrow ◽  
Double Mutant ◽  
Molecular Mechanisms ◽  
Mutant Mice ◽  
Mrna Levels ◽  
Wild Type ◽  
Erythroid Progenitors ◽  
Ineffective Erythropoiesis ◽  
Qrt Pcr

β-thalassemia arises as a result of mutations in the β-globin gene. As a consequence erythropoiesis, the process that insures the daily generation of billions of red blood cells (RBCs), becomes disrupted. Ineffective erythropoiesis is a major contributor to the β-thalassemic anemia and is partially due to aberrant apoptosis during late stages of erythroid maturation. Despite the importance of apoptosis, the underlying molecular mechanisms regulating this process in β-thalassemia erythroblasts are not fully elucidated. One potential mechanism involves the transcription factor Foxo3, which under specific contexts can act as a positive regulator of apoptosis, but is also an essential transcriptional regulator of terminal erythroblast maturation. Foxo3 has a range of outputs that it can execute from sustaining cellular integrity by mitigating oxidative stress to inducing apoptosis under conditions of overwhelming stress. Given these functions, we sought to determine if Foxo3 played a role in maintaining RBC maturation in β-thalassemic mice. To address this, we used Hbbth3/+ (th3/+) mice that display a phenotype similar to β-thalassemia intermedia, and produced double mutant Foxo3-/-/Th3/+ mice. The th3/+ mice display a mild erythroblast apoptotic phenotype. We hypothesized that loss of Foxo3 may exacerbate the β-thalassemic phenotype. On the contrary, we found that loss of Foxo3 in a β-thalassemic background improved RBC numbers and hemoglobin concentration (by 1g/dl, n=10 mice) in double mutant mice compared to th3/+ mice. Furthermore, double mutant mice had a statistically significant lower frequency of apoptosis (2 fold less) during bone marrow erythroblast maturation as measured by flow cytometry analysis of annexin V-binding and 7AAD staining in distinct erythroblast stages resolved by TER119, CD44 and cell size (n=3 mice per genotype). We predicted that high levels of oxidative stress may prematurely activate FOXO3 during erythroblast maturation in β-thalassemic mice. In turn, activated FOXO3 may potentially promote apoptosis in these cells. To evaluate this, we examined FOXO3 levels by qRT-PCR and immunofluorescence in FACS sorted populations of erythroblasts (TER119+,CD44,FSC) or erythroid progenitors (TER119-,c-KIT+,CD71HI) acquired from bone marrow of at least 3 mice per genotype. Our data show increased mRNA levels of Foxo3 in early erythroblasts, corresponding to increased FOXO3 protein expression in erythroid progenitors from β-thalassemic mice relative to wild-type mice. We also examined the activation status of p53, as it is also a major regulator of apoptosis that can be triggered by oxidative stress. Nuclear p53 levels were greater in β-thalassemic as compared to wild-type erythroid progenitors based on immunofluorescence analysis of sorted cells from bone marrow of 3 mice per genotype. These results suggest a higher level of active p53 in β-thalassemic erythroid progenitors. Our results provide evidence that FOXO3, a factor normally critical for erythroblast maturation, may cooperate with aberrantly active p53 to induce apoptosis in β-thalassemic erythroblasts. In support of this, downstream p53 targets including Gadd45a and p21 that are also Foxo3 targets were significantly upregulated in β-thalassemic erythroblasts relative to wild-type erythroblasts as determined by qRT-PCR of cDNA produced from 3 mice per genotype. To more closely examine the mechanism of decreased apoptosis in double mutant Foxo3-/-/Th3/+ erythroblasts, we compared the expression of multiple genes involved in apoptosis by qRT-PCR of sorted erythroblast populations from at least 3 mice per genotype. We found multiple pro-apoptotic genes including, Cycs, Tnfsf10, Puma, and Bim expressed at significantly lower levels at various erythroblast stages in double mutant compared to β-thalassemic erythroblasts. Together, our data suggests Foxo3 becomes inappropriately and prematurely activated in erythroid progenitors and early erythroblasts in the context of β-thalassemia and cooperates with p53 to promote apoptosis. These findings raise the possibility that cooperation of Foxo3 and p53 in β-thalassemic erythroblasts might contribute to the ineffective erythropoiesis of β-thalassemic mice. They also suggest the possibility that as a homeostatic maintaining factor, Foxo3 behaves differently in the context of disease. Disclosures No relevant conflicts of interest to declare.

High-Vorticity with Periodic Flow Enhances in Vitro Biogenesis of Healthy Platelets from iPSC-Derived-Megakaryocytes

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2181-2181
Author(s):  
Yukitaka Ito ◽  
Sou Nakamura ◽  
Tomohiro Shigemori ◽  
Naoshi Sugimoto ◽  
Yoshikazu Kato ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Ex Vivo ◽  
Annexin V ◽  
Poor Quality ◽  
Flow Chamber ◽  
Physical Parameters ◽  
Platelet Production

Abstract Each transfusion requires 200-300 billion platelets in patients with thrombocytopenia. To continuously supply such a huge number of platelets by ex vivo generation, two distinct steps, megakaryopoiesis and platelet shedding, must be both considered. For the former, one approach is to increase the number of source cell, megakaryocytes. For example, the immortalized megakaryocyte cell line (imMKCL) system uses self-renewing megakaryocyte (MK) cell lines derived from induced pluripotent stem cells (iPSCs) (Nakamura et al., Cell Stem Cell, 2014). For the latter, there have been an idea of bioreactors whereby shedding of platelets from proplatelets could be promoted by flow-dependent shear force within the bone marrow in vivo (Junt et al., Science, 2007; Zhang et al., J Exp Med, 2012). Based upon this idea, we constructed a flow chamber type bioreactor recapitulating in vivo blood flow shear rate. However, this bioreactor failed to efficiently yield platelets, and moreover, the produced platelets had poor quality as indicated by high Annexin V levels (Exp Hematol, 2011 and unpublished result). Recently, we demonstrated two different kinetics of platelet biogenesis from bone marrow MKs, whereby either thrombopoietin (TPO) mostly regulates steady-state shedding of platelets from proplatelets, or interleukin-a (IL-1a) triggers inflammation-dependent rupture of MK cytoplasm contributing to a quick increase of platelet count at higher rate (Nishimura et al., J Cell Biol, 2015). However, the rupture type platelets revealed shorter half-life with relatively higher Annexin V levels. Therefore, to gain insights from platelet biogenesis in vivo, we focused on biophysical analysis of steady-state platelet biogenesis via proplatelets in bone marrow. Our observations strongly indicated that the presence of 'vorticity' defined by vortex turbulence in addition to shear-dependent 'stress' and 'strain' correlates with the efficient shedding of competent platelets. From this new finding, we developed an alternative bioreactor system, which enabled generation of 100 billion platelets from imMKCL in a 16L-scale liquid culture condition without any adherent machinery using two 10L-bioreactors. Furthermore, platelets generated via new bioreactors showed low Annexin V levels (<10-15%) and shortened bleeding time post transfusion into NOG mice and rabbits with thrombocytopenia, comparable to human blood product platelets. Regarding the platelet production using WAVE bag system (GE healthcare, UK), the system is already clinically available for cord blood cell expansion in most countries, but lacks adequate levels of vorticity and shear strain/stress. Accordingly, the produced platelets had high Annexin V levels (i.e., 50-65%) as well as diminished yield efficiency (P<0.001). In conclusion, our study has uncovered the novel biophysical aspect of platelet biogenesis. The application of the new set of physical parameters in constructing large sized bioreactors shall facilitate the industrialization of platelet production. Disclosures Eto: Megakaryon Co. Ltd.: Research Funding.

A novel role for PECAM-1 in megakaryocytokinesis and recovery of platelet counts in thrombocytopenic mice

Blood ◽  
2007 ◽  
Vol 109 (10) ◽  
pp. 4237-4244 ◽  
Author(s):  
Tarvinder S. Dhanjal ◽  
Caroline Pendaries ◽  
Ewan A. Ross ◽  
Mark K. Larson ◽  
Majd B. Protty ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Spatial Localization ◽  
Matrix Proteins ◽  
Platelet Production ◽  
Stromal Microenvironment ◽  
Proplatelet Formation ◽  
Stromal Cell Derived Factor ◽  
Deficient Mice

Abstract During thrombopoiesis, maturing megakaryocytes (MKs) migrate within the complex bone marrow stromal microenvironment from the proliferative osteoblastic niche to the capillary-rich vascular niche where proplatelet formation and platelet release occurs. This physiologic process involves proliferation, differentiation, migration, and maturation of MKs before platelet production occurs. In this study, we report a role for the glycoprotein PECAM-1 in thrombopoiesis. We show that following induced thrombocytopenia, recovery of the peripheral platelet count is impaired in PECAM-1–deficient mice. Whereas MK maturation, proplatelet formation, and platelet production under in vitro conditions were unaffected, we identified a migration defect in PECAM-1–deficient MKs in response to a gradient of stromal cell–derived factor 1 (SDF1), a major chemokine regulating MK migration within the bone marrow. This defect could be explained by defective PECAM-1−/− MK polarization of the SDF1 receptor CXCR4 and an increase in adhesion to immobilized bone marrow matrix proteins that can be explained by an increase in integrin activation. The defect of migration and polarization was confirmed in vivo with demonstration of altered spatial localization of MKs within the bone marrow in PECAM-1–deficient mice, following immune-induced thrombocytopenia. This study identifies a novel role for PECAM-1 in regulating MK migration and thrombopoiesis.

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