Role of Fas / FasL in Regulation of Basophillic Erythroblast Homeostasis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4289-4289
Author(s):  
Renold J. Capocasale ◽  
Dorie A. Makropoulos ◽  
Jeffrey Arlen ◽  
Ram Achuthanandam ◽  
John Quinn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fas Ligand ◽  
Annexin V ◽  
Fold Increase ◽  
Similar Process ◽  
Becton Dickinson ◽  
Color Flow ◽  
Deficient Mice

Abstract Many studies have shown Fas- Fas Ligand (FasL) mediated apoptosis to be important in maturation and differentiation of erythroid precursors in vitro. To determine if there is a similar process regulating erythropoietic homeostasis in vivo, we studied erythropoiesis in Fas(lpr) and FasL (gld) deficient mice. We postulated that deficiency of Fas or FasL should result in changes in red blood cell (RBC) parameters and/or decreased levels of apoptosis of erythroblasts. To test this hypothesis under steady state conditions, blood and bone marrow were collected from 10-week old C57Bl/6 control mice, B6.MRL-Tnfrsf6 lpr /J CD95 deficient mice, and B6Smn.C3-Tnfsf6 gld /J CD95L deficient mice. Hematology was studied using a Bayer Advia 120 and femoral bone marrow was analyzed by 6-color flow cytometry using a Becton Dickinson FACSAria. Hematologic analysis revealed no differences in reticulocyte counts, RBC counts or hemoglobin (Hgb) in either lpr or gld mice compared to C57Bl/6 controls. Similarly, analysis of bone marrow revealed no differences in % of Ter-119bright CD71bright basophilic erythroblast (BEB), % apoptotic BEB (annexin V+, 7-AADdim) or % FasL+ BEB in either gld or lpr mice compared to control. As expected, lpr mice expressed 10 fold fewer Fas+ BEB while similar levels were observed in gld mice compared to controls. To test our hypothesis under stimulated conditions, control, lpr and gld mice received a single s.c. dose of 10,000 units of recombinat human erythropoietin (rhEPO). Bone marrow samples were collected 48 hours after dosing and blood samples 4, 8 and 16 days after dosing. Hematologic analysis revealed no differences in the erythropoietic response among the three strains of mice tested. Moreover, treatment with rhEPO had no effect on % Fas+ BEB in any strain, but induced a 2–5 fold increase in the % FasL+ BEB and a 2–3 fold increase in apoptotic BEB in all three strains. Based on our observations, we conclude Fas/FasL is unlikely to play a pivotal role in regulating erythroid homeostasis.

Gene transfer into normal human hematopoietic cells using in vitro and in vivo assays

Blood ◽  
1991 ◽  
Vol 78 (3) ◽  
pp. 624-634 ◽  
Author(s):  
JE Dick ◽  
S Kamel-Reid ◽  
B Murdoch ◽  
M Doedens
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Hematopoietic Cells ◽  
Human Stem Cells ◽  
In Vivo Assays ◽  
Genetically Manipulated ◽  
Deficient Mice

Abstract The ability to transfer new genetic material into human hematopoietic cells provides the foundation for characterizing the organization and developmental program of human hematopoietic stem cells. It also provides a valuable model in which to test gene transfer and long-term expression in human hematopoietic cells as a prelude to human gene therapy. At the present time such studies are limited by the absence of in vivo assays for human stem cells, although recent descriptions of the engraftment of human hematopoietic cells in immune-deficient mice may provide the basis for such an assay. This study focuses on the establishment of conditions required for high efficiency retrovirus- mediated gene transfer into human hematopoietic progenitors that can be assayed in vitro in short-term colony assays and in vivo in immune- deficient mice. Here we report that a 24-hour preincubation of human bone marrow in 5637-conditioned medium, before infection, increases gene transfer efficiency into in vitro colony-forming cells by sixfold; interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) provide the same magnitude increase as 5637-conditioned medium. In contrast, incubation in recombinant growth factors IL-1, IL-3, and granulocyte- macrophage colony-stimulating factor increases gene transfer efficiency by 1.5- to 3-fold. Furthermore, preselection in high concentrations of G418 results in a population of cells significantly enriched for G418- resistant progenitors (up to 100%). These results, obtained using detailed survival curves based on colony formation in G418, have been substantiated by directly detecting the neo gene in individual colonies using the polymerase chain reaction. Using these optimized protocols, human bone marrow cells were genetically manipulated with a neo retrovirus vector and transplanted into immune-deficient bg/nu/xid mice. At 1 month and 4 months after the transplant, the hematopoietic tissues of these animals remained engrafted with genetically manipulated human cells. More importantly, G418-resistant progenitors that contained the neo gene were recovered from the bone marrow and spleen of engrafted animals after 4 months. These experiments establish the feasibility of characterizing human stem cells using the unique retrovirus integration site as a clonal marker, similar to techniques developed to elucidate the murine stem cell hierarchy.

scholarly journals The coordinated action of G-CSF and ELR + CXC chemokines in neutrophil mobilization during acute inflammation

Blood ◽  
2008 ◽  
Vol 111 (1) ◽  
pp. 42-49 ◽  
Author(s):  
Antje M. Wengner ◽  
Simon C. Pitchford ◽  
Rebecca C. Furze ◽  
Sara M. Rankin
Keyword(s):  
Bone Marrow ◽  
Intraperitoneal Injection ◽  
Fold Increase ◽  
In Situ Perfusion ◽  
Acute Peritonitis ◽  
Femoral Bone Marrow ◽  
Prior Administration

In this study, we have identified a unique combinatorial effect of the chemokines KC/MIP-2 and the cytokine granulocyte colony-stimulating factor (G-CSF) with respect to the rapid mobilization of neutrophils from the bone marrow in a model of acute peritonitis. At 2 hours following an intraperitoneal injection of thioglycollate, there was a 4.5-fold increase in blood neutrophil numbers, which was inhibited 84% and 72% by prior administration of blocking mAbs against either the chemokines KC/MIP-2 or G-CSF, respectively. An intraperitoneal injection of G-CSF acted remotely to stimulate neutrophil mobilization, but did not elicit recruitment into the peritoneum. Further, in vitro G-CSF was neither chemotactic nor chemokinetic for murine neutrophils, and had no priming effect on chemotaxis stimulated by chemokines. Here, we show that, in vitro and in vivo, G-CSF induces neutrophil mobilization by disrupting their SDF-1α–mediated retention in the bone marrow. Using an in situ perfusion system of the mouse femoral bone marrow to directly assess mobilization, KC and G-CSF mobilized 6.8 × 106 and 5.4 × 106 neutrophils, respectively, while the infusion of KC and G-CSF together mobilized 19.5 × 106 neutrophils, indicating that these factors act cooperatively with respect to neutrophil mobilization.

External PS in Sickle RBC: Relationships among Aminophospholipid Translocase (APLT), Phospholipid Scramblase (PLSCR), Cell Maturity, and Cell Density.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 148-148
Author(s):  
Latorya E. Arnold ◽  
Mary B. Palascak ◽  
Clinton H. Joiner ◽  
Robert S. Franco
Keyword(s):  
Annexin V ◽  
Type I ◽  
Type Ii ◽  
Color Flow ◽  
Low Level ◽  
Aminophospholipid Translocase ◽  
Phospholipid Scramblase ◽  
High Level

Abstract External phosphatidylserine (PS) is present on some sickle RBC and may contribute to thrombogenesis, endothelial adhesion, and shortened RBC lifespan. Phospholipid scramblase (PLSCR) disrupts phospholipid (PL) asymmetry by causing nonspecific PL equilibration across the membrane. Aminophospholipid translocase (APLT) maintains PL asymmetry by returning externalized PS to the inner membrane leaflet. It has been proposed that both APLT inhibition and PLSCR activation are required for PS externalization. Sickle RBC with low level external PS (Type I PS+) are present in cells of all densities and include some reticulocytes. Sickle RBC with high external PS (Type II PS+) are primarily found in the dense fraction. Type II cells are thought to be more important because: the high level of external PS should have greater consequence; high level external PS occurs primarily in pathologically dehydrated sickle RBC; and low level external PS appears to be physiological in immature RBC. We have previously shown that dense, dehydrated sickle RBC, including the small number of dense transferrin receptor positive (TfR+) reticulocytes, have markedly inhibited APLT. In the current studies, we examined the relationships among external PS, APLT, PLSCR, and density in mature RBC and TfR+ reticulocytes using 3-color flow cytometry. APLT and PLSCR activities were assayed using fluorescent PL analogues (NBD-PS and NBD-PC, respectively), and expressed as the fraction of probe internalized. External PS was measured with Annexin V-PE and TfR+ reticulocytes were identified with anti-TfR-PE/Cy5. PS+ cells had lower APLT activity compared to PS- cells that did not reach significance for n=3 (NBD-PS internalization fraction for PS-: 0.586±0.053; Type I PS+: 0.517±0.158, Type II PS+: 0.523±0.033). PS- sickle RBC had a uniformly low PLSCR activity similar to normal RBC (NBD-PC internalization fractions ∼ 0.1). In mature sickle RBC, PLSCR was more active in PS+ cells (PS-: 0.097±0.096; Type I PS+: 0.163±0.070, Type II PS+: 0.248±0.043; n=3; PS- vs Type I PS+: p=0.06; PS- vs Type II PS+: p=0.04; Type I versus Type II: p=0.03). TfR+ reticulocytes had increased APLT and PLSCR activity compared to mature sickle RBC, but there was no apparent relationship between PLSCR and external PS. Since dense sickle RBC had markedly inhibited APLT, we evaluated the relationship between dehydration and APLT activity. Dehydration of AA RBC from an MCHC of 35.6±2.2 to 49.2±2.0 g/dL inhibited APLT (from 0.484±0.068 to 0.301±0.076; n=7, p= 0.01). Dehydration of SS RBC from an MCHC of 34.8±3.5 to 50.1±3.9 g/dL also inhibited APLT (from 0.460±0.060 to 0.361±0.047; n=3, p=0.006), but not as low as in SS RBC dehydrated in vivo (0.222±0.036 at 44.7±5.6 g/dL; n=4, p=0.007 vs. SS RBC dehydrated in vitro). Rehydration of AA and SS RBC that had been dehydrated in vitro reversed APLT inhibition. However, APLT activity was not reversed upon rehydration of sickle RBC dehydrated in vivo. In summary, our data show that: many dense sickle RBC with significantly inhibited APLT are PS-, indicating that APLT inhibition alone does not result in PS externalization; dehydration contributes to, but is not entirely responsible for, the APLT inhibition seen in dense sickle RBC; and PS+ sickle RBC have increased PLSCR activity.

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.

Combined Fludarabine, Cyclophosphamide, and Alemtuzumab (FCC), an Active Regimen for Treated Patients with Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3133-3133 ◽  
Author(s):  
Marco Montillo ◽  
Sara Miqueleiz ◽  
Alessandra Tedeschi ◽  
Francesca Ricci ◽  
Eleonora Vismara ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Residual Disease ◽  
Single Agent ◽  
Lymphocytic Leukemia ◽  
Combination Regimen ◽  
Synergistic Activity ◽  
Color Flow ◽  
Grade Iii

Abstract Fludarabine (F) in combination with cyclophosphamide (C) showed a relevant advantage over single-agent F in pts with relapsed CLL. Although minimal residual disease (MRD) remains detectable in many pts achieving CR, the combination of F and C seems to reduce MRD more efficiently. Still, pts in CR eventually relapse and require treatment, demonstrating the need for improved treatments able to further reduce or eliminate MRD and induce “better quality” and thus more durable responses. Alemtuzumab (CAM), anti-CD52 monoclonal antibody, acts synergistically with F in vitro and appears to have synergistic activity in vivo. Additionally, CAM is highly effective at clearing disease from bone marrow, the usual site of residual disease following purine analogue-based treatment. Therefore, we designed a phase II study to determine feasibility and efficacy, overall response rate (ORR)-duration of response-ability at clearing MRD, of a 4-weekly combination regimen consisting of F, C, and CAM (FCC). The study population is represented by pts with B-CLL with relapsed or refractory disease after at least one line of treatment. Subcutaneous route of administration of CAM has been adopted in this trial. MRD was measured by 4-color flow cytometry in the bone marrow. The FCC regimen consisted of F 40 mg/m2/d os (d 1–3), C 250 mg/m2/d os (d 1–3) and CAM 10 mg sc (d 1–3). This combination was repeated on d 29 for up to 6 cycles. The dose of CAM was increased after the first cohort of 10 treated pts from 10 mg to 20 mg sc. Currently, 25 pts have been enrolled in this trial. Median age was 57 years (range 42–79), 15/25 (60%) were male, 23/25 (92%) were in Binet stage B or C, median number of prior treatment regimens was 2 (range 1–4). In six (24%) pts 17p deletion was detected. IgVH unmutated was observed in 17 (68%) pts. At the moment of writing 19 pts are eligible for evaluation of toxicity and response. The ORR was 79%, with 7 (37%) pts achieving CR, 7 (37%) pts a PR, 1 (5%) pt a PRn. Three pts had SD, while 1 showed progression of the disease. MRD negativity was achieved in the bone marrow of 4/15 (27%) pts. Grade III-IV neutropenia episodes were observed in 43% of the administered courses while grade III-IV thrombocytopenia episodes were detected only in 8% of cycles. Four major infections were recorded: two sustained by Mycobacterium tuberculosis (1 cutis, 1 lung), one by Nocardia (lung) and one by E. coli (sepsis). The patient with pneumonia due to M. tuberculosis died because of respiratory failure. CMV reactivation occurred in 6 pts: no CMV disease was recorded. After a median follow up of 10 m (range 1–22) 73% of responding pts did not progressed. In conclusion, results from the interim analysis of this new, 4-weekly dosing FCC regimen suggest that combination therapy with F, C and CAM is feasible, safe, and effective in treating pts with relapsed and refractory CLL, even in those patients with inherent poor prognostic factors and who had received.

scholarly journals ASXL2 Is Recurrently Mutated in t(8;21) AML and Regulates Hematopoietic Development

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1051-1051
Author(s):  
Vikas Madan ◽  
Lin Han ◽  
Norimichi Hattori ◽  
Anand Mayakonda ◽  
Qiao-Yang Sun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Research Funding ◽  
Hematopoietic Differentiation ◽  
Wild Type ◽  
Flow Cytometric ◽  
Deficient Mice

Abstract Chromosomal translocation t(8;21) (q22;q22) leading to generation of oncogenic RUNX1-RUNX1T1 fusion is a cytogenetic abnormality observed in about 10% of acute myelogenous leukemia (AML). Studies in animal models and recent next generation sequencing approaches have suggested cooperativity of secondary genetic lesions with t(8;21) in inducing leukemogenesis. In this study, we used targeted and whole exome sequencing of 93 cases (including 30 with matched relapse samples) to profile the mutational landscape of t(8;21) AML at initial diagnosis and post-therapy relapse. We identified recurrent mutations of KIT, TET2, MGA, FLT3, NRAS, DHX15, ASXL1 and KMT2Dgenes in this subtype of AML. In addition, high frequency of truncating alterations in ASXL2 gene (19%) also occurred in our cohort. ASXL2 is a member of mammalian ASXL family involved in epigenetic regulation through recruitment of polycomb or trithorax complexes. Unlike its closely related homolog ASXL1, which is mutated in several hematological malignancies including AML, MDS, MPN and others; mutations of ASXL2 occur specifically in t(8;21) AML. We observed that lentiviral shRNA-mediated silencing of ASXL2 impaired in vitro differentiation of t(8;21) AML cell line, Kasumi-1, and enhanced its colony forming ability. Gene expression analysis uncovered dysregulated expression of several key hematopoiesis genes such as IKZF2, JAG1, TAL1 and ARID5B in ASXL2 knockdown Kasumi-1 cells. Further, to investigate implications of loss of ASXL2 in vivo, we examined hematopoiesis in Asxl2 deficient mice. We observed an age-dependent increase in white blood cell count in the peripheral blood of Asxl2 KO mice. Myeloid progenitors from Asxl2 deficient mice possessed higher re-plating ability and displayed altered differentiation potential in vitro. Flow cytometric analysis of >1 year old mice revealed increased proportion of Lin-Sca1+Kit+ (LSK) cells in the bone marrow of Asxl2 deficient mice, while the overall bone marrow cellularity was significantly reduced. In vivo 5-bromo-2'-deoxyuridine incorporation assay showed increased cycling of LSK cells in mice lacking Asxl2. Asxl2 deficiency also led to perturbed maturation of myeloid and erythroid precursors in the bone marrow, which resulted in altered proportions of mature myeloid populations in spleen and peripheral blood. Further, splenomegaly was observed in old ASXL2 KO mice and histological and flow cytometric examination of ASXL2 deficient spleens demonstrated increased extramedullary hematopoiesis and myeloproliferation compared with the wild-type controls. Surprisingly, loss of ASXL2 also led to impaired T cell development as indicated by severe block in maturation of CD4-CD8- double negative (DN) population in mice >1 year old. These findings established a critical role of Asxl2 in maintaining steady state hematopoiesis. To gain mechanistic insights into its role during hematopoietic differentiation, we investigated changes in histone marks and gene expression affected by loss of Asxl2. Whole transcriptome sequencing of LSK population revealed dysregulated expression of key myeloid-specific genes including Mpo, Ltf, Ngp Ctsg, Camp and Csf1rin cells lacking Asxl2 compared to wild-type control. Asxl2 deficiency also caused changes in histone modifications, specifically H3K27 trimethylation levels were decreased and H2AK119 ubiquitination levels were increased in Asxl2 KO bone marrow cells. Global changes in histone marks in control and Asxl2 deficient mice are being investigated using ChIP-Sequencing. Finally, to examine cooperativity between the loss of Asxl2 and RUNX1-RUNX1T1 in leukemogenesis, KO and wild-type fetal liver cells were transduced with retrovirus expressing AML1-ETO 9a oncogene and transplanted into irradiated recipient mice, the results of this ongoing study will be discussed. Overall, our sequencing studies have identified ASXL2 as a gene frequently altered in t(8;21) AML. Functional studies in mouse model reveal that loss of ASXL2 causes defects in hematopoietic differentiation and leads to myeloproliferation, suggesting an essential role of ASXL2 in normal and malignant hematopoiesis. *LH and NH contributed equally Disclosures Ogawa: Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

Survivin Is Required for Proliferation, but Not Polyploidization of Megakaryocytes.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1328-1328
Author(s):  
Jeremy Q Wen ◽  
Cindy Leung ◽  
Zan Huang ◽  
Sara Small ◽  
John Crispino
Keyword(s):  
Bone Marrow ◽  
Ex Vivo ◽  
Survivin Expression ◽  
Annexin V ◽  
Retroviral Infection ◽  
Hematopoietic Stem ◽  
Significant Difference ◽  
Megakaryocyte Progenitors

Abstract Survivin is a member of chromosome passenger complex, which plays an important role in chromosome alignment, separation and cytokinesis. We recently reported that survivin is necessary for the proliferation and survival of hematopoietic stem and progenitor cells. Furthermore, we previously showed that reduced levels of survivin expression facilitates megakaryocyte development, whereas elevated levels of survivin inhibit their maturation and polyploidization. However, the extent to which survivin is necessary for polyploidization and terminal differentiation of committed megakaryocytes remains unclear. To determine whether survivin is required for megakaryocyte and platelet biogenesis, we mated mice with floxed alleles of survivin (sur fl/fl) to mice that express Cre recombinase under the control of the PF4 promoter. Compound mutant animals appeared grossly normal and harbored normal platelet counts. Furthermore, survivin deleted and control littermates displayed similar expression of CD41 and CD42, as well as similar DNA content within the CD41+ population. The only significant difference detected was an increase in annexin V staining of CD41+ cells within the bone marrow of the mice with survivin deletion. Analysis of DNA extracted from these bone marrows showed no evidence of the survivin deletion, indicating that the surviving cells all escaped excision. These in vivo findings are consistent with a requirement for survivin in the survival or proliferation of megakaryocyte progenitors. Next, to induce megakaryocyte development ex vivo, we cultured bone marrow from surv fl/fl mice in vitro in the presence of TPO. Using this approach, we were able to induce survivin deletion in 75% of the cells as evidenced by PCR. Despite the deletion of survivin, polyploidization of the ex vivo generated megakaryocytes was unaffected. Finally, we induced deletion of survivin by retroviral infection of surv fl/fl progenitors with MSCV-Cre and found that megakaryocyte polyploidization was actually increased in the excised population. Taken together, our results suggest that survivin is not required for polyploidization, but is necessary for proliferation of megakaryocyte progenitors.

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.

Reduced thrombus stability in mice lacking the α2A-adrenergic receptor

Blood ◽  
2006 ◽  
Vol 108 (2) ◽  
pp. 510-514 ◽  
Author(s):  
Miroslava Požgajová ◽  
Ulrich J. H. Sachs ◽  
Lutz Hein ◽  
Bernhard Nieswandt
Keyword(s):  
Blood Flow ◽  
Adrenergic Receptor ◽  
Thrombus Formation ◽  
Fold Increase ◽  
Wild Type ◽  
Perfusion Studies ◽  
G Protein Coupled ◽  
Deficient Mice

Platelet activation plays a central role in hemostasis and thrombosis. Many platelet agonists function through G-protein–coupled receptors. Epinephrine activates the α2A-adrenergic receptor (α2A) that couples to Gz in platelets. Although α2A was originally cloned from platelets, its role in thrombosis and hemostasis is still unclear. Through analysis of α2A-deficient mice, variable tail bleeding times were observed. In vitro, epinephrine potentiated activation/aggregation responses of wild-type but not α2A-deficient platelets as determined by flow cytometry and aggregometry, whereas perfusion studies showed no differences in platelet adhesion and thrombus formation on collagen. To test the in vivo relevance of α2A deficiency, mice were subjected to 3 different thrombosis models. As expected, α2A-deficient mice were largely protected from lethal pulmonary thromboembolism induced by the infusion of collagen/epinephrine. In a model of FeCl3-induced injury in mesenteric arterioles, α2A–/– mice displayed a 2-fold increase in embolus formation, suggesting thrombus instability. In a third model, the aorta was mechanically injured, and blood flow was measured with an ultrasonic flow probe. In wild-type mice, all vessels occluded irreversibly, whereas in 24% of α2A-deficient mice, the initially formed thrombi embolized and blood flow was reestablished. These results demonstrate that α2A plays a significant role in thrombus stabilization.

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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