scholarly journals KIFC1 Mediates Centrosome Clustering Prior to Proplatelet Formation from Megakaryocytes

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 721-721
Author(s):  
Adrian R. Wilkie ◽  
Leila J. Noetzli ◽  
Genevieve Couldwell ◽  
Anthony R. Sciaudone ◽  
Anjana Ray ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Western Blot ◽  
Cancer Cells ◽  
Cell Biology ◽  
Research Funding ◽  
Fetal Liver ◽  
Cell Cortex ◽  
Mouse Bone Marrow ◽  
Platelet Production ◽  
Proplatelet Formation

Platelets are specialized anucleate cells that circulate in the blood and serve to prevent bleeding and minimize blood vessel injury. In addition to their hemostatic functions, platelets participate in wound healing, angiogenesis, inflammation, and immunity, and are therefore central players in both maintaining normal physiology and in disease pathogenesis. Platelets are derived from their precursor cells, megakaryocytes (MKs), that reside principally in the bone marrow. During maturation, MKs undergo an altered cell cycle called endomitosis in which they replicate their DNA but avoid cell division, resulting in polyploid MKs with amplified microtubule (MT)-organizing centers called centrosomes. Subsequently, MT-dependent forces are responsible for extending long cytoplasmic protrusions called proplatelets into sinusoidal blood vessels, eventually giving rise to circulating platelets. Despite progress in elucidating key steps of platelet production, there is a conspicuous lack of understanding of what triggers mature, polyploid MKs to undergo the MT rearrangements required for proplatelet production. Using live cell imaging of mouse fetal liver-derived MKs expressing fluorescent b1-tubulin, we have identified a novel MT-based structure in MKs termed a monospindle. Our data suggest that monospindles result from polyploid MKs clustering multiple centrosomes into a centralized MT-organizing center during mitosis, leading to an enlarged array of MTs oriented towards the cell cortex. These structures were also apparent in mouse bone marrow- and human cord blood-derived MKs, suggesting that monospindle formation is a general phenomenon in MKs. Interestingly, a higher percentage of MKs contained monospindles at a timepoint directly prior to proplatelet production (50%) compared to when proplatelets were actively being produced (22%), indicating a possible role in initiating proplatelet formation. Centrosome clustering in cancer cells is mediated by the MT-based mitotic motor protein, KIFC1. Consistently, we found that small molecule inhibition of KIFC1 decreased the percentage of MKs containing monospindles (55% ctrl vs. 6% KIFC1 inhibitor). Strikingly, KIFC1 inhibitor treatment also drastically reduced the percentage of MKs producing proplatelets (peak proplatelet formation: 40% ctrl vs. 5% KIFC1 inhibitor), suggesting that KIFC1-mediated centrosome clustering into monospindles is important for proplatelet production. To test how KIFC1 contributes to these phenotypes, we assessed its expression at different timepoints by Western blot and detected increased KIFC1 levels in more mature MKs preceding proplatelet formation. Cell sorting of MKs into distinct ploidy populations followed by Western blot showed that KIFC1 expression increased with higher ploidy. Thus, our results lead us to suggest a working model in which elevated KIFC1 levels in mature MKs drive monospindle formation to trigger proplatelet formation. Investigating the role of KIFC1-mediated monospindle formation for initiating proplatelet formation could yield a coherent, molecular understanding of how mature, polyploid MKs reorganize MTs for proplatelet production. In addition, these data will help inform basic cell biology, as there are important parallels between centrosome clustering in MKs and cancer cells. Finally, our findings could yield novel therapeutic strategies for treating patients with thrombocytopenia (low platelet counts) by directly stimulating platelet production from mature MKs residing in the bone marrow. Disclosures Italiano: Platelet Biogenesis: Employment, Equity Ownership; Ionis Research Funding: Research Funding.

scholarly journals Efficient retroviral gene transfer to purified long-term repopulating hematopoietic stem cells

Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 74-83 ◽  
Author(s):  
SJ Szilvassy ◽  
S Cory
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Cell Biology ◽  
Bone Marrow Cells ◽  
High Titer ◽  
Marker Gene ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem

Abstract Efficient gene delivery to multipotential hematopoietic stem cells would greatly facilitate the development of effective gene therapy for certain hematopoietic disorders. We have recently described a rapid multiparameter sorting procedure for significantly enriching stem cells with competitive long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil (5-FU)-treated mouse bone marrow. The sorted cells have now been tested as targets for retrovirus-mediated delivery of a marker gene, NeoR. They were cocultured for 4 days with fibroblasts producing a high titer of retrovirus in medium containing combinations of the hematopoietic growth factors interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) and then injected into lethally irradiated recipients, together with sufficient “compromised” bone marrow cells to provide short-term support. Over 80% of the transplanted mice displayed high levels (> or = 20%) of donor- derived leukocytes when analyzed 4 to 6 months later. Proviral DNA was detected in 87% of these animals and, in half of them, the majority of the hematopoietic cells were marked. Thus, infection of the stem cells was most effective. The tissue and cellular distribution of greater than 100 unique clones in 55 mice showed that most sorted stem cells had lymphoid as well as myeloid repopulating potential. Secondary transplantation provided strong evidence for infection of very primitive stem cells because, in several instances, different secondary recipients displayed in their marrow, spleen, thymus and day 14 spleen colony-forming cells the same proviral integration pattern as the primary recipient. Neither primary engraftment nor marking efficiency varied for stem cells cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower secondary engraftment potential. Provirus expression was detected in 72% of the strongly marked mice, albeit often at low levels. Highly efficient retroviral marking of purified lymphomyeloid repopulating stem cells should enhance studies of stem cell biology and facilitate analysis of genes controlling hematopoietic differentiation and transformation.

Final Results from an International, Multi-Center, Single-Arm Study Evaluating the Safety and Efficacy of Romiplostim in Adults with Primary Immune Thrombocytopenia (ITP),

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3279-3279 ◽  
Author(s):  
Ann Janssens ◽  
Michael D. Tarantino ◽  
Robert Bird ◽  
Maria Gabriella Mazzucconi ◽  
Ralph Vincent V. Boccia ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Board Of Directors ◽  
Research Funding ◽  
Platelet Response ◽  
Employment Equity ◽  
Advisory Committees ◽  
Platelet Production ◽  
Platelet Counts ◽  
Equity Ownership

Abstract Abstract 3279 Background: ITP is an autoimmune disorder characterized by increased platelet destruction and suboptimal platelet production. Romiplostim stimulates platelet production via the TPO-receptor, and is recommended for second- and third-line treatment of chronic ITP in adults. We report final data from a large prospective study of romiplostim in adults with ITP of varying duration and severity. Methods: Eligibility criteria were broad: patients ≥18 years of age, who had received prior ITP therapies (final protocol amendment: ≥1, previous amendments: ≥3), with low platelet counts (final amendment: ≤ 30 × 109/L, previous amendments: ≤ 10, ≤ 20 × 109/L) or experiencing uncontrolled bleeding. The only excluded comorbidities were: hematological malignancy, myeloproliferative neoplasms, MDS and bone marrow stem cell disorder. Romiplostim was initiated at 1 (final amendment) or 3 (previous amendments) μg/kg/week, with dose adjustments allowed to maintain platelet counts ≥50 × 109/L. Patients could continue on study until they had access to commercially available romiplostim. Rescue medications were allowed at any time; concurrent ITP therapies could be reduced when platelet counts were > 50 × 109/L. Primary endpoint was incidence of adverse events (AEs) and antibody formation. Secondary endpoint was platelet response, defined as either (1) doubling of baseline count and ≥ 50 × 109/L or (2) ≥20 × 109/L increase from baseline. Results: A total of 407 patients received romiplostim, 60% of whom were female. Median (Q1, Q3) time since ITP diagnosis was 4.25 (1.20, 11.40) years (maximum 57.1 years), with 51% of patients splenectomised and 39% receiving baseline concurrent ITP therapies. Seventy-one percent of patients completed the study, with requirement for alternative therapy and withdrawn consent the most common reasons for discontinuation (5% each). Median (Q1, Q3) on-study treatment duration was 44.29 (20.43, 65.86) weeks (maximum 201 weeks), with a total of 20,201 subject-weeks on study. Incidence and type of AEs were consistent with previous studies. The most common serious treatment-related AEs were cerebrovascular accident, headache, bone marrow reticulin fibrosis (with no evidence of positive trichrome staining for collagen and no evidence suggesting primary idiopathic myelofibrosis), nausea, deep vein thrombosis, hemorrhage and pulmonary embolism, with each reported in 2 of 407 (0.5%) patients. All other serious treatment-related AEs were each reported in one patient. Eighteen patients died; 3 deaths (hemolysis, intestinal ischaema, aplastic anemia) were considered treatment-related. No neutralizing antibodies to romiplostim or TPO were reported. Approximately 90% of patients achieved each of the platelet response definitions, regardless of splenectomy status. Overall, median (Q1, Q3) time to response was 2 (1, 4) weeks for response definition 1, and 1 (1, 3) week for response definition 2. Median (Q1, Q3) baseline platelet count was 14 (8, 21) × 109/L. After 1 week of treatment median (Q1, Q3) platelet count had increased to 42 (18, 101) × 109/L. From week 8 onwards, and excluding counts within 8 weeks of rescue medication use, median platelet counts were consistently above 100 × 109/L (range 101.0–269.5 × 109/L). Median (Q1, Q3) average weekly romiplostim dose was 3.62 (1.99, 6.08) μg/kg. Summary/conclusions: This is the largest prospective study in adult ITP reported to date. The data reported here are similar to those reported for previous romiplostim studies, with romiplostim able to safely induce a rapid platelet response in adult ITP patients with low platelet counts or bleeding symptoms. Romiplostim is an important, well-tolerated, treatment option for adult ITP patients, which significantly increases and maintains platelet counts. Adverse Event Subject Incidence Platelet Response Disclosures: Janssens: Amgen: Consultancy; Roche: Speakers Bureau; GSK: Membership on an entity's Board of Directors or advisory committees. Tarantino:Cangene corporation: Research Funding; Baxter: Research Funding; Talecris: Honoraria, Speakers Bureau; Up-to-date: Patents & Royalties; The Bleeding and Clotting Disorders Institute: Board Member. Bird:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GSK: Membership on an entity's Board of Directors or advisory committees. Boccia:Amgen: Equity Ownership, Honoraria, Speakers Bureau. Lopez-Fernandez:Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Kozak:Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Steurer:Amgen: Honoraria. Dillingham:Amgen Limited: Employment, Equity Ownership. Lizambri:Amgen: Employment, Equity Ownership.

scholarly journals Effects of 12-0-tetradecanoylphorbol-13-acetate (TPA) on the proliferation of granulocyte-macrophage colony-forming cells

Blood ◽  
1983 ◽  
Vol 61 (3) ◽  
pp. 575-579 ◽  
Author(s):  
AW Burgess ◽  
NA Nicola
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Mouse Bone Marrow ◽  
Gm Csf ◽  
Accessory Cells ◽  
Macrophage Colony ◽  
Stimulating Factor ◽  
Csf Production ◽  
Number Of Cells

Abstract It has been suggested that 12–0-tetradecanoylphorbol-13-acetate (TPA) may stimulate the proliferation of granulocyte-macrophage (GM) colony- forming cells (CFC) via the GM colony-stimulating factor (CSF) receptor. GM-CFC in unfractionated mouse bone marrow and light density fetal liver (LDFL) cells were induced by TPA to form colonies in the absence of exogenously added GM-CSF. The colonies induced by TPA (10(- 8)M) were smaller than normally seen with maximal concentrations of GM- CSF, and less than 30% of the GM-CFC formed colonies in the presence of TPA. The number of colonies stimulated by TPA in the absence of GM-CSF was dependent on the number of cells plated. When fewer than 10,000 bone marrow cells or 3000 LDFL cells were plated in the 1-ml semisolid agar cultures, no colonies were stimulated by the TPA. Similarly, GM- CFC purified from the LDFL cells stimulated with TPA did not form colonies. However, when the fetal liver accessory cells (macrophages) were recombined with cell-sorter-purified GM-CFC, colony formation was again observed in the presence of TPA (10(-7)-10(-8) M). The number of colonies formed from the CFC was dependent on the number of accessory cells present, suggesting that the macrophages were induced by TPA to produce CSF. Although the purified GM-CFC required CSF for proliferation, TPA (10(-8) M) increased (5–10-fold) the sensitivity of the GM-CFC to GM-CSF. These observations indicate that TPA does not stimulate GM-CFC proliferation directly, but rather by inducing GM-CSF production by accessory cells and by increasing the responsiveness of GM-CFC to GM-CSF.

scholarly journals Identification and characterization of osteoclast progenitors by clonal analysis of hematopoietic cells

Blood ◽  
1992 ◽  
Vol 80 (7) ◽  
pp. 1710-1716 ◽  
Author(s):  
MY Lee ◽  
JL Lottsfeldt ◽  
KL Fevold
Keyword(s):  
Bone Marrow ◽  
Tumor Cell ◽  
Mononuclear Cells ◽  
Fetal Liver ◽  
Tumor Cell Line ◽  
Adherent Cell ◽  
Mouse Bone Marrow ◽  
Hematopoietic Progenitors ◽  
Adult Mice

Abstract We have identified a distinct population of colony-forming cells that give rise to mononuclear cells expressing an enzyme marker and other features of the osteoclast in bone marrow cultures stimulated by conditioned medium of a murine tumor cell line. These colony-forming cells were defined as osteoclast colony-forming units (CFU-O). The tumor cell-derived activity was recently isolated and was named osteoclast colony-stimulating factor (O-CSF). To understand the development of osteoclast progenitors and to clarify the relationship of osteoclast progenitors to other hematopoietic progenitors, we examined CFU-O in hematopoietic tissues obtained from normal adult mice, mouse fetuses, and mice with 5-fluorouracil (5FU) treatment. CFU- O were present in the adult mouse bone marrow, adherent cell-depleted marrow, in the spleen, and in the day 14 fetal liver, with an incidence similar to other hematopoietic progenitors. The culture period required for the development of CFU-O-derived colonies in vitro and the manner in which CFU-O responded to 5FU suggested that CFU-O belonged to a relatively primitive progenitor population; they are clearly more immature than macrophage progenitors that respond to macrophage-CSF, but more mature than multilineage progenitors that respond to stem cell factor. Our studies have defined and characterized an osteoclast progenitor and distinguished it from other hematopoietic progenitors for the first time.

Development Of 3D Models For Studying Megakaryopoiesis In Vitro

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3696-3696
Author(s):  
Lindsay Wray ◽  
Christian A Di Buduo ◽  
David L. Kaplan ◽  
Alessandra Balduini
Keyword(s):  
Mechanical Properties ◽  
Bone Marrow ◽  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Peripheral Blood ◽  
Platelet Production ◽  
Vascular Niche ◽  
Proplatelet Formation ◽  
Bone Marrow Vascular Niche

Abstract Introduction Silk fibroin, derived from Bombyx mori silkworm cocoons, is used extensively in biomaterials and regenerative medicine. The useful characteristics of this protein include self-assembly, robust mechanical properties, biocompatibility and biodegradability. Moreover, silk can be enhanced through a variety of chemical modifications that affect cell attachment, growth and differentiation. Thrombocytopenia occurs when a patient suffers from an abnormally low platelet count in the peripheral blood; usually a result of disease, trauma, or cancer treatment. To treat these patients, it is estimated that two million platelet transfusions are performed in the U.S. each year. This high demand for platelets has created a clinical demand for studying the causes of thrombocytopenia and alternative routes for treatment. Platelets are anuclear cells that are released into the bloodstream in the bone marrow by megakaryocytes via the extension of long filaments called proplatelets. It is hypothesized that platelet production from megakaryocytes is regulated by environmental factors at the site of bone marrow vascular niche. Studies of megakaryopoiesis are typically performed on extracellular matrix protein-coated culture plates and transwell membranes. While these initial studies have provided invaluable insight into the process of megakaryopoiesis, the goal of the present project was to create a bone marrow model that mimics the vascular niche for functional in vitro platelet production. We hypothesized that a silk-based in vitro tissue model would allow the effects of substrate surface properties and endothelial co-culture on megakaryopoiesis to be studied in a holistic manner, thereby enabling further elucidation of the mechanisms involved in the process of platelet production. Results In order to more closely mimic the bone marrow vascular niche structure, a porous silk sponge was assembled around the silk vessel-like tubes. Megakarycytes seeded in the porous silk sponge migrated toward the silk tube and released platelets into the tube lumen. The perfusion bioreactor moved the platelets into the platelet collecting bags. After perfusion the platelets were collected and analyzed by flow cytometry. The bioreactor platelets exhibited similar morphology, CD41 positive staining, and activation compared to peripheral blood platelet controls. Megakaryocyte attachment and proplatelet formation through the silk vascular wall were improved by altering the silk properties. Silk functionalized by entrapping extracellular matrix proteins within the tube membrane resulted in increased megakaryocyte attachment and proplatelet compared to unfunctionalized silk tube controls. Silk surface roughness improved megakaryocyte attachment compared to the control but did not affect proplatelets. Decreasing the silk stiffness improved proplatelets, but did not significantly affect megakaryocyte attachment. Co-culture with endothelial cells improved megakaryocyte attachment while maintaining a high level of proplatelet formation. Additionally, megakaryocyte and endothelial cell co-culture on the silk vessel model resulted in an icreased platelet production compared to megakaryocytes cultured alone. Conclusions The goal of this project was to develop an in vitro model of megakaryopoiesis using a tissue engineering approach. Using human megakaryocytes and endothelial cells, we demonstrate the following advanced features of the silk-based model: (1) immobilization of extracellular matrix components within the membrane, (2) tunable surface topography, (3) tunable mechanical properties, (4) physiologically relevant thickness for appropriate proplatelet extension, and (5) controlled localization of a vascular endothelium. Thus, by functionalizing silk, we can control megakaryocyte function on silk. The broader impact of this work offers a versatile new tool for studying megakaryocyte development and platelet production in vitro. Disclosures: No relevant conflicts of interest to declare.

Small Molecule PRIMA-1 met Sensitizes Waldenstrom Macroglobulinemia Cells To Apoptosis and Displays Synergistic Cytotoxicity With Bortezomib

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5143-5143
Author(s):  
Mona Sobhani ◽  
Kim Kwan ◽  
Manujendra N. Saha ◽  
Christine I. Chen ◽  
Hong Chang
Keyword(s):  
Bone Marrow ◽  
Western Blot ◽  
Cell Viability ◽  
Small Molecule ◽  
Mtt Assay ◽  
Research Funding ◽  
Mutant P53 ◽  
Haematological Malignancies ◽  
Wild Type ◽  
Qrt Pcr

Abstract Background Waldenstrom macroglobulinemia (WM) is a lymphoplasmacytic lymphoma characterized by heterogeneous infiltration of bone marrow and IgM monoclonal gammopathy. WM constitutes 1-2% of haematological malignancies and patients exhibit diverse outcome. Given the current therapies WM remains incurable with high rate of relapse, therefore, the quest for finding a more effective drug continues. PRIMA-1met is a small molecule with the ability to restore wild type conformation to mutant P53 and to activate wild type P53. PRIMA-1met has shown significant anti-tumour activity both in vitro and in vivo by inducing apoptosis in several human cancers including haematological malignancies and is currently under phase I/II clinical trials. To date, the effects of PRIMA-1met have not been explored in WM. Here we examined the antitumor activity of PRIMA-1met alone and in combination with dexamethasone or bortezomib in WM. Design WM cell lines, BCWM-1(Wild type P53) and MWCL-1 (mutant P53), were used for these studies. Cellular proliferation, viability, migration and percentage of cells undergoing apoptosis in WM cells treated with PRIMA-1met or DMSO control were evaluated using trypan blue, MTT assay, Boyden chamber assay and Annexin-V staining methods respectively. The status of key role players in apoptosis and cell cycle regulators were examined following treatment with PRIMA-1met using qRT-PCR and Western blot analyses. Cell viability was also investigated by combining PRIMA-1metwith either dexamethasone or bortezomib in WM cells by MTT assay. Result Both BCWM-1 and MWCL-1 cells showed significant decrease in cell viability after exposure to escalating doses of PRIMA-1met with observed IC50 of 30 μM in BCWM-1 cells and 28 μM in MWCL-1 cells after 48 hours. Two primary WM patients’ samples exposed to increasing concentration of PRIMA-1met demonstrated significant decrease in cell viability with IC50 of 30 and 10 μM after 48 hours. Importantly, peripheral and bone marrow mononuclear cells of 3 healthy donors were subjected to PRIMA-1met and no significant cytotoxicity was observed. Both cell lines showed an increase in apoptosis after PRIMA-1met treatment in a dose-dependent manner compared to control DMSO treatment. In BCWM-1, cell proliferation, measured by counting viable cells after 7 days, was significantly inhibited by PRIMA-1met compared to DMSO control (∼54% fewer cells when treated with 25 μM PRIMA-1met, p<0.05). Decrease in cell migration by as much as 28% at 25 μM compared to DMSO control (p<0.001) was also observed after 8 hours of treatment with PRIMA-1met. Expressions of P53 and its downstream target p21 were elevated in PRIMA-1met treated cells compared to DMSO control. In addition, qRT-PCR studies of 50 genes associated with apoptosis also demonstrated enhanced expression of pro-apoptotic markers such as PUMA, Noxa, caspase 8 and 9, BIM and BAD, which were further confirmed for their protein expressions by Western blot analysis. Finally, PRIMA-1met (25 μM) was found to exert synergistic cytotoxic effect in combination with dexamethasone (2 μM) in MWCL-1 cells (Combination index (CI)=0.5). PRIMA-1met (25 μM) also displayed synergism with bortezomib (3 μM) in its cytotoxicity toward BCWM-1 cells (CI=0.8). Conclusions Our results demonstrate that PRIMA-1met induces apoptosis irrespective of P53 status in WM cells. It also provides the preclinical framework for evaluation of PRIMA-1met either alone or in combination with bortezomib or dexamethasone as a novel therapeutic approach for the treatment of WM patients. Disclosures: Chen: Roche: Honoraria; Johnson & Johnson: Consultancy, Research Funding; Lundbeck: Consultancy; Celgene: Consultancy, Research Funding; GlaxoSmithKline: 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.

scholarly journals Mouse fetal liver cells lack functional amphotropic retroviral receptors

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 433-439 ◽  
Author(s):  
C Richardson ◽  
M Ward ◽  
S Podda ◽  
A Bank
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Retroviral Vectors ◽  
Liver Cells ◽  
Mouse Bone Marrow ◽  
Adult Mice ◽  
Fetal Liver Cells ◽  
Marrow Cells

Abstract We have been transducing mouse hematopoietic cells with the human MDR1 (MDR) gene in retroviral vectors to determine the optimal conditions for retroviral gene transfer as a model system for potential human gene therapy. In these studies, we have demonstrated transduction and expression of the human MDR gene using ecotropic and amphotropic MDR- retroviral producer lines. To obtain more mouse hematopoietic cells for detailed study, mouse fetal liver cells (FLC) have been used for MDR transduction and expression, and to reconstitute the ablated marrows of live adult mice. FLC contain hematopoietic cells that have a reconstituting capacity comparable to that of adult mouse bone marrow cells. However, to our surprise, FLC can only be transduced with ecotropic retrovirus and not with amphotropic virus. This restriction of transduction of FLC cannot be overcome by higher titer virus. The resistance to amphotropic transduction by FLC may be part of a changing developmental program that results in a different antigen repertoire on FLC as compared with adult bone marrow cells.

scholarly journals Improved Patient-Derived Xenograft Model for Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3491-3491 ◽  
Author(s):  
Xiaochuan Shan ◽  
Cedric Dos Santos ◽  
Chenghui Zhou ◽  
Georges Habineza Ndikuyeze ◽  
Anthony Secreto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
Xenograft Model ◽  
Mouse Strains ◽  
Mouse Bone Marrow ◽  
Tissue Samples ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Nsg Mice ◽  
Patient Derived Xenograft

Abstract We previously demonstrated that the NSG (NOD-Scid-IL2Rgcnull) xenotransplantation is an improved model for human AML samples, allowing us to better understand and characterize AML biology, especially in the context of drug therapy studies (Sanchez et al., Leukemia 2009). However, we observed that approximately half of AML patients’ samples either did not engraft in NSG mice (based on <0.1% human blasts in mouse bone marrow) or showed low (0.1 to 1% blasts) and highly variable engraftment. Recently, NSG mice expressing human SCF, GM-CSF and IL-3 transgenes (NSG-S) have been reported to enhance the engraftment of normal hematopoietic stem cells and primary AML cells, although only a few AML patients were evaluated (Wunderlich M et al, Leukemia 2010). This report describes a comprehensive paired analysis of engraftment of AML samples in NSG and NSG-S mice. T-cell depleted AML cells (5 -10 x 106 per mouse) were injected intravenously in sub-lethally irradiated mice (n=5/AML sample). Leukemia engraftment was assessed up to 16 weeks after injection in peripheral blood (PB), spleen (SPL) and bone marrow (BM) based on the percentage and absolute number of human leukemic blasts (huCD45+CD33+/-CD3-) in each tissue. Samples from 71 AML patients, representing all FAB and prognosis groups, were injected in NSG mice and only 35 samples (49%) engrafted based on human blasts >0.5% in mouse BM. From these 35 NSG-engrafting samples, 14 were also injected in NSG-S mice and all of them engrafted. Leukemic burden was significantly (p ≤ 0.05) increased in NSG-S versus NSG mice: 39±21% vs 22±23% BM blasts, 21±15% vs 7±10% SPL blasts, 2,732±6,488 vs 141±221 blasts/ml PB. Interestingly, the dramatic increase in peripheral blast count observed in NSG-S mice provides new opportunities to use PB to monitor drug response for the many patient samples that show no or very low peripheral engraftment in NSG mice. However, for 7 of these 14 NSG-engrafting AML samples, the use of NSG-S mice as recipients was associated with rapid engraftment, excessive leukemic burden, anemia, weight loss and lethargy requiring early sacrifice and leading to shorter overall survival (54±26 days in NSG-S vs >90 days in NSG). Out of the 36 patients’ samples that failed to engraft in NSG mice, 19 were tested for engraftment in NSG-S mice. Remarkably, 14 out 19 (74%) samples engrafted (17±16% BM blasts, 8±12% SPL, and 1,418±4,609/ml PB blasts at Day 77 post-transplant) and the kinetics of engraftment were slower compared to AML samples that can engraft in both mouse strains. These results suggest that the presence of human SCF, GM-CSF and IL-3 in NSG-S is sufficient to rescue leukemia-initiating cells for most AML samples that fail to engraft in NSG mice. Only 5 out of 33 samples (15%) failed to engraft in NSG and NSG-S mice, indicating that the NSG-S BM microenvironment remains suboptimal for a small minority of AML samples. We are investigating if NSG-S engraftment is correlated to CD116, CD117, CD123 expression, cytogenetics, mutations, and prognosis. Overall, our results show that NSG-S mice represent a significant improvement over previous patient-derived xenograft models since they can (1) accelerate and enhance leukemic engraftment compared to NSG mice, and (2) support engraftment for 85% of our AML patients, making this model particularly useful for pre-clinical studies. Disclosures Dos Santos: Janssen R&D: Research Funding. Danet-Desnoyers:Janssen R&D: Research Funding.

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