The Experts below are selected from a list of 240 Experts worldwide ranked by ideXlab platform
Marten Hansen - One of the best experts on this subject based on the ideXlab platform.
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The RNA-Binding Protein ATXN2 is Expressed during Megakaryopoiesis and May Control Timing of Gene Expression
International journal of molecular sciences, 2020Co-Authors: Marten Hansen, Sabrina Zeddies, Marjolein Meinders, Franca Di Summa, Ewa Rollmann, Floris P. J. Van Alphen, Aj Hoogendijk, Kat S. Moore, Melanie Vanessa Halbach, Laura GutierrezAbstract:Megakaryopoiesis is the process during which Megakaryoblasts differentiate to polyploid megakaryocytes that can subsequently shed thousands of platelets in the circulation. Megakaryocytes accumulate mRNA during their maturation, which is required for the correct spatio-temporal production of cytoskeletal proteins, membranes and platelet-specific granules, and for the subsequent shedding of thousands of platelets per cell. Gene expression profiling identified the RNA binding protein ATAXIN2 (ATXN2) as a putative novel regulator of megakaryopoiesis. ATXN2 expression is high in CD34+/CD41+ Megakaryoblasts and sharply decreases upon maturation to megakaryocytes. ATXN2 associates with DDX6 suggesting that it may mediate repression of mRNA translation during early megakaryopoiesis. Comparative transcriptome and proteome analysis on megakaryoid cells (MEG-01) with differential ATXN2 expression identified ATXN2 dependent gene expression of mRNA and protein involved in processes linked to hemostasis. Mice deficient for Atxn2 did not display differences in bleeding times, but the expression of key surface receptors on platelets, such as ITGB3 (carries the CD61 antigen) and CD31 (PECAM1), was deregulated and platelet aggregation upon specific triggers was reduced.
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Generation and characterization of human iPSC lines SANi001-A and SANi002-A from mobilized peripheral blood derived Megakaryoblasts
Stem cell research, 2017Co-Authors: Marten Hansen, Eszter Varga, Tatjana Wüst, Clemens Mellink, Anne-marie Van Der Kevie-kersemaekers, Marieke Von Lindern, Emile Van Den AkkerAbstract:Abstract Mobilized peripheral blood (MPB) CD34 + cells were differentiated to CD34 + /CD41 + Megakaryoblasts. Cells were sorted to obtain a pure Megakaryoblast population which was reprogrammed with a hOKSM self-silencing polycistronic lentiviral vector. Resulting iPSC showed normal karyotype and expression of pluripotency associated markers and in vitro spontaneous differentiation towards the 3 germ layers confirmed pluripotency of iPSC lines. Besides normal iPSC applications, these lines can be used as a control line for other megakaryoid origin iPSC and could be applied for epigenetic based research.
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Generation and characterization of human iPSC line MML-6838-Cl2 from mobilized peripheral blood derived Megakaryoblasts
Stem cell research, 2016Co-Authors: Marten Hansen, Eszter Varga, Tatjana Wüst, Nelleke P.m. Brouwer, Hugues Beauchemin, Clemens Mellink, Anne-marie Van Der Kevie-kersemaekers, Tarik Möröy, Bert A. Van Der Reijden, Marieke Von LindernAbstract:Abstract Mobilized peripheral blood (MPB) CD34 + cells were cultured to CD41 +/CD34 + Megakaryoblasts. Cells were sorted to obtain a pure Megakaryoblast population that was reprogramed by a hOKSM self-silencing polycistronic vector using lentiviral delivery. The generated induced pluripotent stem cell (iPSC) lines were tested for silencing of the reprogramming construct by flow cytometry. Pluripotency of MML-6838-Cl2 iPSC line was confirmed by expression of associated markers and by in vivo spontaneous differentiation towards the 3 germ layers. The genomic integrity of iPSC line was shown by karyotyping. The MML-6838-Cl2 iPSC is, to our knowledge, the first to be generated from Megakaryoblasts.
Mariusz Z. Ratajczak - One of the best experts on this subject based on the ideXlab platform.
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RGS16 “tightens the reins” on CXCR4
Blood, 2005Co-Authors: Mariusz Z. RatajczakAbstract:Comment on Berthebaud et al, page [2962][1] While expression of CXCR4, the receptor for the α-chemokine stromal-derived factor 1 (SDF-1), is relatively high on mature megakaryocytes, these cells lose their responsiveness to stimulation by SDF-1 compared with young Megakaryoblasts. RGS16, a member
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Biological significance of chemokine receptor expression by normal human Megakaryoblasts.
Folia histochemica et cytobiologica, 2001Co-Authors: Marcin Majka, Jacek Kijowski, Ryan Reca, J Ratajczak, Baj-krzyworzek M, Bogusław Machaliński, Kubicze K, Mariusz Z. RatajczakAbstract:The aim of this study was to learn more on the role of chemokines in the regulation of human megakryopoiesis. Normal human Megakaryoblasts were expanded in serum-free liquid cultures and subsequently (1) phenotyped for expression of various chemokine receptors, (2) evaluated if chemokine receptors which they express are functional after stimulation by chemokines (calcium flux assay, chemotaxis, phosphorylation of MAPK-p42/44 and AKT proteins), and (3) investigated for expression and secretion of selected chemokines by employing RT-PCR and ELISA assays, respectively. In addition we also phenotyped peripheral blood platelets for expression of chemokine receptors and chemokines. We found that while human Megakaryoblasts express several chemokine receptors (CXCR4, CCR6, CCR8, CCR5, CCR2 and CXCR3), CXCR4 was the only receptor detectable by FACS on human platelets. Moreover, among various chemokines tested, only SDF-1 (CXCR4 ligand) stimulated calcium flux and chemotaxis in normal human Megakaryoblasts and phosphorylated MAPK-p42/44 and AKT in these cells. Although mRNAs for several chemokines were detectable by RT-PCR in normal human Megakaryoblasts, only RANTES, IL-8, MCP-1 and PF-4 were found to be secreted by these cells. Finally we noticed that no chemokine tested in this study affected CFU-Meg colony formation by human CD34+ cells in serum-free cultures. We conclude that from all the chemokine receptor-chemokine axes tested, only SDF-1-CXCR4 axis was functional in assays employed in our studies, which further support the view that this axis plays a privileged role in regulating normal human megakaryopoiesis.
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In vitro expansion of human megakaryocytes as a tool for studying megakaryocytic development and function.
Platelets, 2001Co-Authors: Marcin Majka, Janina Ratajczak, Monika Baj-krzyworzeka, Jacek Kijowski, Ryan Reca, Mariusz Z. RatajczakAbstract:Research on normal human megakaryopoiesis has been limited by technical problems in obtaining megakaryocytic cells in sufficient quantities for experimental purposes. We describe here an ex vivo serum-free liquid culture system to expand normal human Megakaryoblasts from purified bone marrow-, cord blood- or peripheral blood-derived CD34(+) cells. The early megakaryocytic cells are expanded in the presence of recombinant thrombopoietin (TpO) and interleukin-3 (IL-3), and if necessary further purified by employing anti-CD61 immunomagnetic beads. Our expansion system generates normal human Megakaryoblasts in quantities sufficient to perform various functional studies on these cells as well as to isolate from them proteins and mRNA for molecular analysis. Megakaryocytic cells isolated from these cultures (i) express several markers characteristic of this lineage (CD41, CD61, CD62 P, CXCR-4, PAR-1, etc.), (ii) respond by calcium flux and phosphorylation of various intracellular proteins to stimulation by thrombin and (iii) adhere to fibrinogen and vitronectin. However, human Megakaryoblasts derived from the cultures supplemented with TpO + IL-3, in contrast to murine megakaryocytic cells cultured under similar conditions, display poor polyploidization and do not release platelets. Since IL-3 has been reported to inhibit final maturation of megakaryocytic cells, we recently modified our expansion strategy. In this new approach CD34(+) cells are first expanded for 11 days in the presence of TpO + IL-3. Then Megakaryoblasts derived are expanded for an additional 7 days supplemented with TpO only. We found that megakaryocytic cells expanded in this 'two step culture' model are more differentiated, are polyploid and release platelets. The model described here provides normal human Megakaryoblasts in adequate numbers, to study megakaryopoiesis and megakaryocyte function.
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Binding of stromal derived factor‐1α(SDF‐1α) to CXCR4 chemokine receptorin normal human Megakaryoblasts butnot in platelets induces phosphorylationof mitogen‐activated protein kinase p42/44 (MAPK), ELK‐1 transcription factor and serine/threonine kina
European journal of haematology, 2000Co-Authors: Marcin Majka, Janina Ratajczak, M. Anna Kowalska, Mariusz Z. RatajczakAbstract:The aim of this study was to identify pathways which are involved in signal transduction from the CXCR4 receptor stimulated by stromal derived factor-1alpha (SDF-1alpha) in human malignant hematopoietic cells and normal Megakaryoblasts. First, we found that activation of CXCR4 in human T cell lines (Jurkat and ATL-2) rapidly induced phosphorylation of mitogen-activated protein kinases (MAPK) (p44 ERK-1 and p42 ERK-2). Next, we became interested in CXCR4-mediated signaling in normal hematopoietic cells, and employed human Megakaryoblasts, which highly express CXCR4 as a model. We found that stimulation of these cells with SDF-1alpha led to the phosphorylation of MAPK and serine/threonine kinase AKT as well. Activation of MAPK further led to the phosphorylation of the nuclear transcription factor ELK-1. Phosphorylation of ELK-1 in Megakaryoblasts implies that phosphorylated MAPK translocate from cytoplasm into the nucleus where they may phosphorylate some nuclear proteins. Note that neither MAPK nor AKT was phosphorylated in normal human platelets after stimulation by SDF-1. We conclude that both MAPK and AKT are involved in signal transduction pathways from the CXCR4 receptor in malignant and normal human hematopoietic cells. The biological consequences of MAPK, ELK-1 and AKT phosphorylation in Megakaryoblasts after stimulation with SDF-1alpha require further studies.
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Bone marrow CD34(+) cells and Megakaryoblasts secrete beta-chemokines that block infection of hematopoietic cells by M-tropic R5 HIV.
The Journal of clinical investigation, 1999Co-Authors: Marcin Majka, Tomasz Rozmyslowicz, Benhur Lee, Samuel L. Murphy, Zbigniew Pietrzkowski, Glen N. Gaulton, Leslie E. Silberstein, Mariusz Z. RatajczakAbstract:CD34+ cells are nonpermissive to infection by HIV strains X4 and R5, despite the fact that many CD34+ cells express high levels of the viral receptor protein CD4 and the coreceptor CXCR4 on their surface. In these cells, the co-receptor CCR5 protein, which, like CXCR4, is a chemokine receptor, is detected mainly intracellularly. We hypothesized that CD34+ cells secrete CCR5-binding chemokines and that these factors interfere with HIV R5 interactions with these cells, possibly by binding CCR5 or by inducing its internalization. We found that human CD34+ cells and CD34+KIT+ cells, which are enriched in myeloid progenitor cells, expressed and secreted the CCR5 ligands RANTES, MIP-1α, and MIP-1β and that IFN-γ stimulated expression of these chemokines. In contrast, SDF-1, a CXCR4 ligand, was not detectable in the CD34+KIT+ cells, even by RT-PCR. Conditioned media from CD34+ cell culture significantly protected the T lymphocyte cell line PB-1 from infection by R5 but not X4 strains of HIV. Interestingly, the secretion of endogenous chemokines decreased with the maturation of CD34+ cells, although ex vivo, expanded Megakaryoblasts still secreted a significant amount of RANTES. Synthesis of CCR5-binding chemokines by human CD34+ cells and Megakaryoblasts therefore largely determines the susceptibility of these cells to infection by R5 HIV strains. We postulate that therapeutic agents that induce the endogenous synthesis of chemokines in human hematopoietic cells may protect these cells from HIV infection. J. Clin. Invest. 104:1739–1749 (1999).
Marcin Majka - One of the best experts on this subject based on the ideXlab platform.
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In vitro expansion of human megakaryocytes as a tool for studying megakaryocytic development and function.
Platelets, 2001Co-Authors: Marcin Majka, Janina Ratajczak, Monika Baj-krzyworzeka, Jacek Kijowski, Ryan Reca, Mariusz Z. RatajczakAbstract:Research on normal human megakaryopoiesis has been limited by technical problems in obtaining megakaryocytic cells in sufficient quantities for experimental purposes. We describe here an ex vivo serum-free liquid culture system to expand normal human Megakaryoblasts from purified bone marrow-, cord blood- or peripheral blood-derived CD34(+) cells. The early megakaryocytic cells are expanded in the presence of recombinant thrombopoietin (TpO) and interleukin-3 (IL-3), and if necessary further purified by employing anti-CD61 immunomagnetic beads. Our expansion system generates normal human Megakaryoblasts in quantities sufficient to perform various functional studies on these cells as well as to isolate from them proteins and mRNA for molecular analysis. Megakaryocytic cells isolated from these cultures (i) express several markers characteristic of this lineage (CD41, CD61, CD62 P, CXCR-4, PAR-1, etc.), (ii) respond by calcium flux and phosphorylation of various intracellular proteins to stimulation by thrombin and (iii) adhere to fibrinogen and vitronectin. However, human Megakaryoblasts derived from the cultures supplemented with TpO + IL-3, in contrast to murine megakaryocytic cells cultured under similar conditions, display poor polyploidization and do not release platelets. Since IL-3 has been reported to inhibit final maturation of megakaryocytic cells, we recently modified our expansion strategy. In this new approach CD34(+) cells are first expanded for 11 days in the presence of TpO + IL-3. Then Megakaryoblasts derived are expanded for an additional 7 days supplemented with TpO only. We found that megakaryocytic cells expanded in this 'two step culture' model are more differentiated, are polyploid and release platelets. The model described here provides normal human Megakaryoblasts in adequate numbers, to study megakaryopoiesis and megakaryocyte function.
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Biological significance of chemokine receptor expression by normal human Megakaryoblasts.
Folia histochemica et cytobiologica, 2001Co-Authors: Marcin Majka, Jacek Kijowski, Ryan Reca, J Ratajczak, Baj-krzyworzek M, Bogusław Machaliński, Kubicze K, Mariusz Z. RatajczakAbstract:The aim of this study was to learn more on the role of chemokines in the regulation of human megakryopoiesis. Normal human Megakaryoblasts were expanded in serum-free liquid cultures and subsequently (1) phenotyped for expression of various chemokine receptors, (2) evaluated if chemokine receptors which they express are functional after stimulation by chemokines (calcium flux assay, chemotaxis, phosphorylation of MAPK-p42/44 and AKT proteins), and (3) investigated for expression and secretion of selected chemokines by employing RT-PCR and ELISA assays, respectively. In addition we also phenotyped peripheral blood platelets for expression of chemokine receptors and chemokines. We found that while human Megakaryoblasts express several chemokine receptors (CXCR4, CCR6, CCR8, CCR5, CCR2 and CXCR3), CXCR4 was the only receptor detectable by FACS on human platelets. Moreover, among various chemokines tested, only SDF-1 (CXCR4 ligand) stimulated calcium flux and chemotaxis in normal human Megakaryoblasts and phosphorylated MAPK-p42/44 and AKT in these cells. Although mRNAs for several chemokines were detectable by RT-PCR in normal human Megakaryoblasts, only RANTES, IL-8, MCP-1 and PF-4 were found to be secreted by these cells. Finally we noticed that no chemokine tested in this study affected CFU-Meg colony formation by human CD34+ cells in serum-free cultures. We conclude that from all the chemokine receptor-chemokine axes tested, only SDF-1-CXCR4 axis was functional in assays employed in our studies, which further support the view that this axis plays a privileged role in regulating normal human megakaryopoiesis.
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Binding of stromal derived factor‐1α(SDF‐1α) to CXCR4 chemokine receptorin normal human Megakaryoblasts butnot in platelets induces phosphorylationof mitogen‐activated protein kinase p42/44 (MAPK), ELK‐1 transcription factor and serine/threonine kina
European journal of haematology, 2000Co-Authors: Marcin Majka, Janina Ratajczak, M. Anna Kowalska, Mariusz Z. RatajczakAbstract:The aim of this study was to identify pathways which are involved in signal transduction from the CXCR4 receptor stimulated by stromal derived factor-1alpha (SDF-1alpha) in human malignant hematopoietic cells and normal Megakaryoblasts. First, we found that activation of CXCR4 in human T cell lines (Jurkat and ATL-2) rapidly induced phosphorylation of mitogen-activated protein kinases (MAPK) (p44 ERK-1 and p42 ERK-2). Next, we became interested in CXCR4-mediated signaling in normal hematopoietic cells, and employed human Megakaryoblasts, which highly express CXCR4 as a model. We found that stimulation of these cells with SDF-1alpha led to the phosphorylation of MAPK and serine/threonine kinase AKT as well. Activation of MAPK further led to the phosphorylation of the nuclear transcription factor ELK-1. Phosphorylation of ELK-1 in Megakaryoblasts implies that phosphorylated MAPK translocate from cytoplasm into the nucleus where they may phosphorylate some nuclear proteins. Note that neither MAPK nor AKT was phosphorylated in normal human platelets after stimulation by SDF-1. We conclude that both MAPK and AKT are involved in signal transduction pathways from the CXCR4 receptor in malignant and normal human hematopoietic cells. The biological consequences of MAPK, ELK-1 and AKT phosphorylation in Megakaryoblasts after stimulation with SDF-1alpha require further studies.
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PI-3k-Akt axis inhibits apoptosis in normal human Megakaryoblasts and is efficiently activated by thrombopoietin
Experimental Hematology, 2000Co-Authors: Marcin Majka, Janina Ratajczak, Alan M. GewirtzAbstract:Abstract While optimizing a clinical protocol for ex vivo expansion of human megakaryocytic progenitor cells (CFU-Meg) we found that megakaryocytic cells gradually undergo apoptosis upon prolonged culture or decrease in optimal concentrations of growth factors. Hence, we become interested in the anti-apoptotic influence of several megakaryopoietic stimulators (TPO, IL-6, IL-11 and SDF-1). Megakaryoblasts were expanded ex vivo in serum free liquid cultures from CD34 + cells. When necessary cells were further purified by immunoselection (final purity >95% of α IIb /β 3 + cells), and then exposed to TPO, IL-6, IL-11 or SDF-1. We focused on i) influence of these factors on survival of megakaryocytic cells and ii) correlation with activation of different intracellular signaling pathways (phosphorylation of AKT, MAPK p42/44 and JAK-STAT proteins) with antiapoptotic effects. We found that TPO only, and not IL-6, IL-11 or SDF-1 i) protected human Megakaryoblasts from undergoing apoptosis (Annexin-V binding, PARP and MTT assays and activation of caspase-3) and ii) activated the JAK-STAT, MAPK p42/44 and PI-3K-AKT pathway (Table). We also found that blocking PI-3K activity with wortmanin or Ly 294002 induced apoptosis in human Megakaryoblasts. In contrast inhibiting MAPK p42/44 activity by PD980092 did not induce apoptosis in these cells. This suggests that the PI-3K-AKT axis plays an important role in inhibiting apoptosis in megakaryocytic cells. Interestingly, while the PI-3K-AKT axis was stimulated in normal human Megakaryoblasts by both TPO and SDF-1, only TPO inhibited apoptosis. We conclude that i) PI-3K-AKT axis plays an important anti-apoptotic role in normal human Megakaryoblasts, and ii) TPO in contrast to SDF-1 efficiently activates pathways downstream from AKT or parallel to the PI-3K-AKT pathway. Activation of this cascade appears to prevent normal human megakaryocytic cells from undergoing apoptosis.
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Bone marrow CD34(+) cells and Megakaryoblasts secrete beta-chemokines that block infection of hematopoietic cells by M-tropic R5 HIV.
The Journal of clinical investigation, 1999Co-Authors: Marcin Majka, Tomasz Rozmyslowicz, Benhur Lee, Samuel L. Murphy, Zbigniew Pietrzkowski, Glen N. Gaulton, Leslie E. Silberstein, Mariusz Z. RatajczakAbstract:CD34+ cells are nonpermissive to infection by HIV strains X4 and R5, despite the fact that many CD34+ cells express high levels of the viral receptor protein CD4 and the coreceptor CXCR4 on their surface. In these cells, the co-receptor CCR5 protein, which, like CXCR4, is a chemokine receptor, is detected mainly intracellularly. We hypothesized that CD34+ cells secrete CCR5-binding chemokines and that these factors interfere with HIV R5 interactions with these cells, possibly by binding CCR5 or by inducing its internalization. We found that human CD34+ cells and CD34+KIT+ cells, which are enriched in myeloid progenitor cells, expressed and secreted the CCR5 ligands RANTES, MIP-1α, and MIP-1β and that IFN-γ stimulated expression of these chemokines. In contrast, SDF-1, a CXCR4 ligand, was not detectable in the CD34+KIT+ cells, even by RT-PCR. Conditioned media from CD34+ cell culture significantly protected the T lymphocyte cell line PB-1 from infection by R5 but not X4 strains of HIV. Interestingly, the secretion of endogenous chemokines decreased with the maturation of CD34+ cells, although ex vivo, expanded Megakaryoblasts still secreted a significant amount of RANTES. Synthesis of CCR5-binding chemokines by human CD34+ cells and Megakaryoblasts therefore largely determines the susceptibility of these cells to infection by R5 HIV strains. We postulate that therapeutic agents that induce the endogenous synthesis of chemokines in human hematopoietic cells may protect these cells from HIV infection. J. Clin. Invest. 104:1739–1749 (1999).
Maria Bai - One of the best experts on this subject based on the ideXlab platform.
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Chronic myelogenous leukemia terminating in acute Megakaryoblastic leukemia. Case report.
Journal of experimental & clinical cancer research : CR, 1998Co-Authors: Repousis P, Stavroula Tsiara, Leonidas Christou, P. Konstantinidou, Maria BaiAbstract:A case of chronic myelogenous leukemia (CML) terminating in acute Megakaryoblastic leukemia (AMKL) is here presented. Megakaryoblasts were identified by the presence of platelet peroxidase in the bone marrow as well as in pleural effusion and ascites. The clinical course, morphology and immunologic studies of the blast cells are described in this report.
Marieke Von Lindern - One of the best experts on this subject based on the ideXlab platform.
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Generation and characterization of human iPSC lines SANi001-A and SANi002-A from mobilized peripheral blood derived Megakaryoblasts
Stem cell research, 2017Co-Authors: Marten Hansen, Eszter Varga, Tatjana Wüst, Clemens Mellink, Anne-marie Van Der Kevie-kersemaekers, Marieke Von Lindern, Emile Van Den AkkerAbstract:Abstract Mobilized peripheral blood (MPB) CD34 + cells were differentiated to CD34 + /CD41 + Megakaryoblasts. Cells were sorted to obtain a pure Megakaryoblast population which was reprogrammed with a hOKSM self-silencing polycistronic lentiviral vector. Resulting iPSC showed normal karyotype and expression of pluripotency associated markers and in vitro spontaneous differentiation towards the 3 germ layers confirmed pluripotency of iPSC lines. Besides normal iPSC applications, these lines can be used as a control line for other megakaryoid origin iPSC and could be applied for epigenetic based research.
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Generation and characterization of human iPSC line MML-6838-Cl2 from mobilized peripheral blood derived Megakaryoblasts
Stem cell research, 2016Co-Authors: Marten Hansen, Eszter Varga, Tatjana Wüst, Nelleke P.m. Brouwer, Hugues Beauchemin, Clemens Mellink, Anne-marie Van Der Kevie-kersemaekers, Tarik Möröy, Bert A. Van Der Reijden, Marieke Von LindernAbstract:Abstract Mobilized peripheral blood (MPB) CD34 + cells were cultured to CD41 +/CD34 + Megakaryoblasts. Cells were sorted to obtain a pure Megakaryoblast population that was reprogramed by a hOKSM self-silencing polycistronic vector using lentiviral delivery. The generated induced pluripotent stem cell (iPSC) lines were tested for silencing of the reprogramming construct by flow cytometry. Pluripotency of MML-6838-Cl2 iPSC line was confirmed by expression of associated markers and by in vivo spontaneous differentiation towards the 3 germ layers. The genomic integrity of iPSC line was shown by karyotyping. The MML-6838-Cl2 iPSC is, to our knowledge, the first to be generated from Megakaryoblasts.