49 results on '"Luc Douay"'
Search Results
2. Comprehensive Proteomic Analysis of Human Erythropoiesis
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Patrick Mayeux, Narla Mohandas, Anna Raimbault, Michael Dussiot, Virginie Salnot, John Hale, Yael Zermati, Sarah Ducamp, Marie-Catherine Giarratana, Luc Douay, François Guillonneau, Marjorie Leduc, Frédérique Verdier, Emilie-Fleur Gautier, Catherine Lacombe, Institut Cochin (IC UM3 (UMR 8104 / U1016)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Plateforme protéomique 3P5 [Institut Cochin] (3P5), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Red Cell Physiology Laboratory [New York, USA], New York Blood Center, Centre de Recherche Saint-Antoine (UMRS893), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Ligue Nationale Contre le Cancer - Paris, Ligue Nationnale Contre le Cancer, ANR-11-IDEX-0005,USPC,Université Sorbonne Paris Cité(2011), Institut Cochin ( UM3 (UMR 8104 / U1016) ), Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratory of Excellence GR-Ex, Sorbonne Paris Cité-Université Paris Descartes - Paris 5 ( UPD5 ) -Imagine Institute, Plateforme protéomique 3P5 [Institut Cochin] ( 3P5 ), Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), New York Blood Center - NYBC, Centre de Recherche Saint-Antoine ( CR Saint-Antoine ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), ANR-11-IDEX-0005-02/11-LABX-0051,GR-Ex,Biogenèse et pathologies du globule rouge ( 2011 ), ANR-11-IDEX-0005-02/11-IDEX-0005,USPC,USPC ( 2011 ), Bos, Mireille, Université Sorbonne Paris Cité - - USPC2011 - ANR-11-IDEX-0005 - IDEX - VALID, and Ligue Nationale Contre le Cancer (LNCC)
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Proteomics ,0301 basic medicine ,Cell type ,Erythroblasts ,Proteome ,Cellular differentiation ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Reticulocyte ,hemic and lymphatic diseases ,medicine ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Humans ,Erythropoiesis ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,RNA, Messenger ,lcsh:QH301-705.5 ,[ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Cells, Cultured ,Erythroid Precursor Cells ,Messenger RNA ,RNA ,Cell Differentiation ,Molecular biology ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,lcsh:Biology (General) - Abstract
SUMMARY Mass spectrometry-based proteomics now enables the absolute quantification of thousands of proteins in individual cell types. We used this technology to analyze the dynamic proteome changes occurring during human erythropoiesis. We quantified the absolute expression of 6,130 proteins during erythroid differentiation from late burst-forming units-erythroid (BFU-Es) to orthochromatic erythroblasts. A modest correlation between mRNA and protein expression was observed. We identified several proteins with unexpected expression patterns in erythroid cells, highlighting a breakpoint in the erythroid differentiation process at the basophilic stage. We also quantified the distribution of proteins between reticulocytes and pyrenocytes after enucleation. These analyses identified proteins that are actively sorted either with the reticulocyte or the pyrenocyte. Our study provides the absolute quantification of protein expression during a complex cellular differentiation process in humans, and it establishes a framework for future studies of disordered erythropoiesis., In Brief Gautier et al. use quantitative mass spectrometry to determine the absolute proteome composition of human erythroid progenitors throughout the differentiation process and the quantitative distribution of proteins between reticulocytes and pyrenocytes after enucleation.
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- 2016
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3. Génération de globules rouges de culture à partir de cellules souches : bref récit du futur
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Luc Douay and Christelle Mazurier
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0301 basic medicine ,Blood transfusion ,business.industry ,medicine.medical_treatment ,Biology ,Phenotype ,Embryonic stem cell ,Regenerative medicine ,General Biochemistry, Genetics and Molecular Biology ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,030220 oncology & carcinogenesis ,medicine ,Personalized medicine ,Stem cell ,Induced pluripotent stem cell ,business ,Adult stem cell - Abstract
Human adult pluripotent stem cells, stem cells of embryonic origin and induced pluripotent stem cells (iPS) provide cellular sources for new promising regenerative medicine approaches. Because these cells can be patient-specific, they allow considering a personalized medicine appropriate to the diagnosis of each. The generation of cultured red blood cells (cRBC) derived from stem cells is emblematic of personalized medicine. Indeed, these cells have the advantage of being selected according to a blood phenotype of interest and they may provide treatments to patients in situation of impossible transfusion (alloimmunized patients, rare phenotypes). Essential progresses have established proof of concept for this approach, still a concept some years ago. From adult stem cells, all steps of upstream research were successfully achieved, including the demonstration of the feasibility of injection into human. This leads us to believe that Red Blood Cells generated in vitro from stem cells will be the future players of blood transfusion. However, although theoretically ideal, these stem cells raise many biological challenges to overcome, although some tracks are identified.
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- 2016
4. Transgene-free hematopoietic stem and progenitor cells from human induced pluripotent stem cells
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Nathalie Chevallier, Loïc Garçon, Laurence Guyonneau-Harmand, Hélène Lapillonne, Luc Douay, Marc Benderitter, Brigitte Birebent, François Delhommeau, Christophe Desterke, Thierry Jaffredo, Bruno Homme, Alain Chapel, Université Pierre et Marie Curie - Paris 6 (UPMC), Laboratoire d'Hématologie et d'Immunologie [CHU Saint-Antoine], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Saint-Antoine [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU), EFS Ile de France, Unité d’Ingénierie et de Thérapie Cellulaire, Créteil, F-94017, France., PRP-HOM/SRBE/LRTE, Institut de Radioprotection et de Sûreté Nucléaire (IRSN), Unité de service de l'Institut André Lwoff (US 33 Inserm), Hôpital Paul Brousse-Institut André Lwoff [Villejuif] (IAL), Biomécanique cellulaire et respiratoire (BCR), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)-Centre National de la Recherche Scientifique (CNRS), CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Centre de Recherche Saint-Antoine (UMRS893), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), CHU Saint-Antoine [AP-HP], Migration et différenciation des cellules souches hématopoiétiques = Migration and differentiation of hematopoietic stem cells (LBD-E06), Laboratoire de Biologie du Développement (LBD), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Service d'hématologie clinique et de thérapie cellulaire [CHU Saint-Antoine], Direction Générale de l’Armement ASTRID/ANR program, Etablissement Français du Sang APR 2013, association 'Combattre La Leucémie', joint grant from Agence Nationale pour la Recherche/California Institute for Regenerative Medicine (ANR/CIRM 0001-02), Laboratoire de Radiopathologie et de Thérapies Expérimentales (IRSN/PRP-HOM/SRBE/LRTE), Unité mixte de service UMS33 [Institut André Lwoff] (UMS33 Inserm/IAL), Hôpital Paul Brousse-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut André Lwoff [Villejuif] (IAL), and Jaffredo, Thierry
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[SDV.MHEP.HEM] Life Sciences [q-bio]/Human health and pathology/Hematology ,Myeloid ,Population ,[SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC] ,Biology ,CXCR4 ,03 medical and health sciences ,0302 clinical medicine ,Directed differentiation ,[SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC] ,medicine ,Progenitor cell ,education ,hemogenic endothelium ,030304 developmental biology ,Hemogenic endothelium ,0303 health sciences ,education.field_of_study ,[SDV.BDD.EO] Life Sciences [q-bio]/Development Biology/Embryology and Organogenesis ,[SDV.MHEP.HEM]Life Sciences [q-bio]/Human health and pathology/Hematology ,[SDV.BBM.MN]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular Networks [q-bio.MN] ,Cell biology ,hematopoietic stem cells ,human induced pluripotent stem cells ,Haematopoiesis ,hematopoietic reconstitution ,medicine.anatomical_structure ,[SDV.BDD.EO]Life Sciences [q-bio]/Development Biology/Embryology and Organogenesis ,[SDV.BBM.MN] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular Networks [q-bio.MN] ,030220 oncology & carcinogenesis ,Immunology ,Homing (hematopoietic) - Abstract
Introductory paragraphThe successful production of Hematopoietic Stem and Progenitor Cells (HSPCs) from human pluripotent sources is conditioned by transgene delivery1-5. We describe here a dedicated and tractable one step, GMP-grade, transgene-free and stroma-free protocol to produce HSPCs from human induced pluripotent stem cells (hiPSCs). This procedure, applied to several sources of hiPSCs with equal efficiency, is based on a directed differentiation with morphogens and cytokines to generate a cell population close to nascent HSPCs or their immediate forerunners i.e., hemogenic endothelial cells6-9. Following engraftment into immunocompromised recipients, this cell population was proved capable of a robust myeloid, lymphoid and definitive red blood cell production in sequential recipients for at least 40 weeks. Further identification of the repopulating cells show that they express the G protein–coupled receptor APELIN (APLNR) and the homing receptor CXCR4. This demonstrates that the generation of bona fide HSPCs from hiPSCs without transgenes is possible and passes through an early endo-hematopoietic intermediate. This work opens the way to the generation of clinical grade HSPCs for the treatment of hematological diseases and holds promise for the analysis of HSPC development in the human species.
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- 2017
5. Design and validation of a consistent and reproducible manufacture process for the production of clinical-grade CD34+ expanded stem cells
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E. Mai, A. Black, Hélène Rouard, S. Derenne, Sandrine Vogt, Anthony Criquet, Philippe Henon, Joe-Elie Salem, Laurence Harmand, Luc Douay, Claire Saucourt, Brigitte Birebent, and A. Chartois-Leauté
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Cancer Research ,Transplantation ,business.industry ,Computer science ,Immunology ,CD34 ,Clinical grade ,Cell Biology ,Oncology ,Scientific method ,Immunology and Allergy ,Production (economics) ,Stem cell ,Process engineering ,business ,Genetics (clinical) - Published
- 2017
6. Patents on Red Blood Cell Manufacturing In Vitro
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Laurence Guyonneau-Harmand and Luc Douay
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Red blood cell ,medicine.anatomical_structure ,Developmental Neuroscience ,Chemistry ,medicine ,Cell Biology ,Molecular biology ,In vitro ,Developmental Biology - Published
- 2011
7. RETRACTED: From Stem Cell to Red Blood Cells In Vitro: 'The 12 Labors of Hercules'
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Luc Douay
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business.industry ,Biochemistry (medical) ,Clinical Biochemistry ,In vitro ,Blood substitute ,Cell biology ,Clinical Practice ,Blood cell ,Haematopoiesis ,Red blood cell ,medicine.anatomical_structure ,medicine ,Stem cell ,business ,Induced pluripotent stem cell - Abstract
This article describes the research in progress that will permit the large-scale production of human red blood cells from hematopoietic stem cells. It also discusses the current state of this research, suggests the obstacles to be overcome to pass from the laboratory model to clinical practice, and analyzes the possible indications in the medium and long term. The potential interest of pluripotent stem cells as an unlimited source of red blood cells is considered. If it succeeds, this new approach could mark a considerable advance in the field of transfusion.
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- 2010
8. Stem cell therapy for the treatment of severe tissue damage after radiation exposure
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Noëlle Mathieu, J. Simon, M. Mothy, Christelle Demarquay, Christine Linard, Jean-Jacques Lataillade, Norbert Claude Gorin, Luc Douay, A. Semont, Alain Chapel, Claire Squiban, and Hélène Rouard
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Cancer Research ,Transplantation ,Pathology ,medicine.medical_specialty ,business.industry ,medicine.medical_treatment ,Immunology ,Cell ,Cell Biology ,Stem-cell therapy ,Radiation exposure ,medicine.anatomical_structure ,Oncology ,Tissue damage ,medicine ,Immunology and Allergy ,business ,Genetics (clinical) - Published
- 2018
9. Molecular signature of erythroblast enucleation in human embryonic stem cells
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Nicolas Hebert, Anne-Marie Faussat, Charles Durand, Marie Cambot, Hélène Lapillonne, Shaghayegh Rouzbeh, Christelle Mazurier, Luc Douay, Ladan Kobari, CHU Saint-Antoine [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Institut National de la Transfusion Sanguine [Paris] (INTS), Cancer, Inflammation, Hormones, Université Pierre et Marie Curie - Paris 6 (UPMC)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Biologie du Développement [IBPS] (LBD), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), CHU Trousseau [APHP], Labex GR-Ex fellowship, Etablissement Francais du Sang (EFS), Fondation pour la recherche medicale, Combattre La Leucemie, DIM Stempole (Paris, France), ANR-11-IDEX-0005,USPC,Université Sorbonne Paris Cité(2011), Laboratoire de Biologie du Développement [Paris] (LBD), Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), HAL-UPMC, Gestionnaire, and Université Sorbonne Paris Cité - - USPC2011 - ANR-11-IDEX-0005 - IDEX - VALID
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Cell type ,Embryonic stem cells ,Erythroblasts ,Enucleation ,Human Embryonic Stem Cells ,Embryoid body ,Biology ,MiR-30a ,Erythroblast ,hemic and lymphatic diseases ,[SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN] ,microRNA ,Humans ,Erythropoiesis ,RNA, Messenger ,Cells, Cultured ,Gene knockdown ,Cell Differentiation ,Cell Biology ,Anatomy ,Embryonic stem cell ,Cell biology ,MicroRNAs ,Gene Expression Regulation ,Molecular Medicine ,[SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN] ,Developmental Biology - Abstract
While enucleation is a critical step in the terminal differentiation of human red blood cells, the molecular mechanisms underlying this unique process remain unclear. To investigate erythroblast enucleation, we studied the erythroid differentiation of human embryonic stem cells (hESCs), which provide a unique model for deeper understanding of the development and differentiation of multiple cell types. First, using a two-step protocol, we demonstrated that terminal erythroid differentiation from hESCs is directly dependent on the age of the embryoid bodies. Second, by choosing hESCs in two extreme conditions of erythroid culture, we obtained an original differentiation model which allows one to study the mechanisms underlying the enucleation of erythroid cells by analyzing the gene and miRNA (miR) expression profiles of cells from these two culture conditions. Third, using an integrated analysis of mRNA and miR expression profiles, we identified five miRs potentially involved in erythroblast enucleation. Finally, by selective knockdown of these five miRs we found miR-30a to be a regulator of erythroblast enucleation in hESCs. Stem Cells 2015;33:2431–2441
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- 2015
10. Ex Vivo Expansion Does Not Alter the Capacity of Umbilical Cord Blood CD34+Cells to Generate Functional T Lymphocytes and Dendritic Cells
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Jean C. Gluckman, Luc Douay, Marie C. Giarratana, Michelle Rosenzwajg, and Ladan Kobari
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Receptors, Antigen, T-Cell, alpha-beta ,T-Lymphocytes ,CD34 ,Antigens, CD34 ,Mice, SCID ,Dendritic cell differentiation ,Biology ,Interferon-gamma ,Mice ,Organ Culture Techniques ,Mice, Inbred NOD ,Animals ,Humans ,Cytotoxic T cell ,Lymphopoiesis ,Antigen-presenting cell ,Cells, Cultured ,Follicular dendritic cells ,Cell Differentiation ,Dendritic Cells ,Cell Biology ,Fetal Blood ,Cell biology ,Phenotype ,Cord blood ,Immunology ,Molecular Medicine ,Stem cell ,Developmental Biology - Abstract
We examined whether ex vivo expansion of umbilical cord blood progenitor cells affected their capacity to generate immune cells such as T lymphocytes (TLs) and dendritic cells (DCs). The capacity to generate TLs from cord blood CD34(+) cells expanded for 14 days (d14) was compared with that of nonexpanded CD34(+) cells (d0) using fetal thymus organ cultures or transfer into nonobese diabetic/severe combined immunodeficient mice. The cell preparations yielded comparable percentages of immature (CD4(+)CD8(-), CD4(+)CD8(+)) TLs and functional mature (CD3(+)CD4(+), CD3(+)CD8(+)) TLs with an analogous TCR (T-cell receptor)-Vbeta repertoire pattern. As regards DCs, d0 and d14 CD34(+) cells also yielded similar percentages of CD1a(+) DCs with the same expression levels of HLA-DR, costimulatory and adhesion molecules, and chemokine receptors. DCs derived from either d14 or d0 CD34(+) stimulated allogeneic TLs to the same extent, and the cytokine pattern production of these allogeneic TLs was similar with no shift toward a predominant Th1 or Th2 response. Even though the intrinsic capacity of d14 CD34(+) cells to generate DCs was 13-fold lower than that of d0 CD34(+) cells, this reduction was offset by the prior amplification of the CD34(+) cells, resulting in the overall production of 15-fold more DCs. These data indicate that ex vivo expansion of CD34(+) cells does not impair T lymphopoiesis nor DC differentiation capacity.
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- 2006
11. Génération in vitro de globules rouges humains matures et fonctionnels : un modèle d’étude aux perspectives multidisciplinaires
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Marie-Catherine Giarratana and Luc Douay
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Blood cell ,Red blood cell ,Haematopoiesis ,medicine.anatomical_structure ,Reticulocyte ,Chemistry ,Cellular differentiation ,medicine ,Erythropoiesis ,Stem cell factor ,General Medicine ,Stem cell ,Cell biology - Abstract
We describe a technical approach permitting massive expansion of CD34+ stem cells (up to 1.95 x 10(6)-fold) and their full ex vivo conversion into mature red blood cells (RBCs). This three-step protocol can be adapted to hematopoietic stem cells (HSC) of various origins. First, cell proliferation and erythroid differentiation are induced in serum-free media supplemented with stem cell factor, interleukin-3 and erythropoietin (Epo) for 8 days. The cells are then co-cultured with either the murine stromal cell line MS-5 or human mesenchymal cells for 3 days in the presence of Epo alone. Finally, all exogenous factors are withdrawn and the cells are incubated on a simple stroma for up to 10 days. The ex vivo microenvironment strongly influences both the terminal maturation of erythroid cells and hemoglobin (Hb) synthesis. Critically, in vitro-generated RBCs have all the characteristics of functional native adult RBCs in terms of their enzyme content, membrane deformability, and capacity to fix and release oxygen. In addition, their behavior in the murine NOD/SCID model mirrors that of native RBCs. This new concept of "cultured RBCs" (cRBC) has major implications for basic research on terminal erythropoiesis and for patient management. Currently, the potential yield of functional red cells is compatible with clinical requirements, as several units of packed RBCs can be produced from a single donation. Importantly, infused cRBC would all have a life-span of about 120 days, whereas the mean half-life of normal donor RBCs is only 28 days. This would help to minimize the transfusion exposure of patients requiring regular treatment, thereby reducing the risk of iron overload and allo-immunization. The use of autologous CD34+ cells isolated from leukapheresis samples could be beneficial for patients who no longer tolerate allogeneic RBCs. This new method should also prove useful for analyzing the mechanisms of terminal erythropoiesis, including hemoglobin synthesis. Finally, it could provide a tool for investigating the lifecycle of blood parasites such as Plasmodium, the agent of malaria.
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- 2005
12. Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells
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Laurent Kiger, Michael C. Marden, David Chalmers, Luc Douay, Ladan Kobari, Hélène Lapillonne, Thérèse Cynober, Marie-Catherine Giarratana, and Henri Wajcman
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Erythrocytes ,Reticulocytes ,Time Factors ,Stromal cell ,Ultraviolet Rays ,Cellular differentiation ,Cell Culture Techniques ,Biomedical Engineering ,CD34 ,Antigens, CD34 ,Bioengineering ,Cell Separation ,Mice, SCID ,Biology ,Applied Microbiology and Biotechnology ,Hemoglobins ,Mice ,Mice, Inbred NOD ,hemic and lymphatic diseases ,medicine ,Animals ,Humans ,Cells, Cultured ,Erythroid Precursor Cells ,Microscopy, Confocal ,Stem Cells ,Cell Differentiation ,Genetic Therapy ,Flow Cytometry ,Hematopoietic Stem Cells ,Coculture Techniques ,Cell biology ,Oxygen ,Haematopoiesis ,Red blood cell ,medicine.anatomical_structure ,Immunology ,Cytokines ,Molecular Medicine ,Erythropoiesis ,Stem cell ,Ex vivo ,Biotechnology - Abstract
We describe here the large-scale ex vivo production of mature human red blood cells (RBCs) from hematopoietic stem cells of diverse origins. By mimicking the marrow microenvironment through the application of cytokines and coculture on stromal cells, we coupled substantial amplification of CD34(+) stem cells (up to 1.95 x 10(6)-fold) with 100% terminal differentiation into fully mature, functional RBCs. These cells survived in nonobese diabetic/severe combined immunodeficient mice, as do native RBCs. Our system for producing 'cultured RBCs' lends itself to a fundamental analysis of erythropoiesis and provides a simple in vitro model for studying important human viral or parasitic infections that target erythroid cells. Further development of large-scale production of cultured RBCs will have implications for gene therapy, blood transfusion and tropical medicine.
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- 2005
13. HOX11L2 expression defines a clinical subtype of pediatric T-ALL associated with poor prognosis
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Paola Ballerini, Maryvonne Busson-Le Coniat, Beatrice Pellegrino, Christine Perot, Mircea Adam, Roland Berger, Jessica Zucman-Rossi, Xin Ying Su, Luc Douay, Annick Blaise, Olivier Bernard, Jacqueline Van Den Akker, and Judith Landman-Parker
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Male ,medicine.medical_specialty ,Leukemia, T-Cell ,Neoplasm, Residual ,Adolescent ,Immunology ,Locus (genetics) ,Biology ,Biochemistry ,Immunophenotyping ,Proto-Oncogene Proteins ,Acute lymphocytic leukemia ,Metalloproteins ,Basic Helix-Loop-Helix Transcription Factors ,medicine ,Humans ,Gene family ,Child ,Cyclin-Dependent Kinase Inhibitor p16 ,T-Cell Acute Lymphocytic Leukemia Protein 1 ,Adaptor Proteins, Signal Transducing ,Chromosome Aberrations ,Homeodomain Proteins ,Oncogene Proteins ,Gene Expression Profiling ,Cytogenetics ,Infant ,Cell Biology ,Hematology ,Gene rearrangement ,LIM Domain Proteins ,Precursor Cell Lymphoblastic Leukemia-Lymphoma ,Prognosis ,medicine.disease ,Survival Analysis ,Minimal residual disease ,Neoplasm Proteins ,DNA-Binding Proteins ,Childhood T Acute Lymphoblastic Leukemia ,Child, Preschool ,Cytogenetic Analysis ,Cancer research ,Female ,Transcription Factors ,TAL1 - Abstract
The most frequent oncogenic activation events characterized in childhood T acute lymphoblastic leukemia (T-ALL) result in the transcriptional activation of genes coding for transcription factors. The main genes are TAL1/SCL, a member of the basic region helix-loop-helix gene family, and HOX11L2, a member of the homeobox-containing protein family. To gain insight into the pathogenesis of this type of hematologic malignancy, we analyzed 28 T-ALL samples. SIL-TAL1/SCL fusion was detected in 6 patients; expression of HOX11L2 was observed in 6 patients and of HOX11 in 3 patients. With one exception, these activations did not occur simultaneously in the same patients, and they allowed the subclassification of 50% of the patients.SIL-TAL1 fusion was detected in association withHOX11 expression in one patient and with a t(8;14) (q24;q11) in another. High expression of LYL1,LMO2, or TAL1 was observed mainly in samples negative for HOX11L2 expression. HOX11L1 andHOX11 expression were observed in one instance each, in the absence of detectable chromosomal abnormality of their respective loci, on chromosomes 2 and 10, respectively. HOX11L2 expression was associated with a chromosome 5q abnormality, the location of theHOX11L2 locus in each case tested. Finally, our data show that HOX11L2 expression was a suitable marker for minimal residual disease follow-up and was significantly associated with relapse (P = .02).
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- 2002
14. Clono-Specific Evaluation of Minimal Residual Disease in Acute Myeloid Leukemia
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Chrystele bilhou Nabera, François Delhommeau, Pierre Hirsch, Ruoping Tang, Ollivier Legrand, Nassera Abermil, Luc Douay, Hannah Moatti, Pascale Flandrin, and Mohamad Mohty
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NPM1 ,Chemotherapy ,medicine.medical_specialty ,Pathology ,IDH1 ,business.industry ,medicine.medical_treatment ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,Biochemistry ,Minimal residual disease ,Gastroenterology ,Regimen ,Internal medicine ,CEBPA ,Cytarabine ,Medicine ,business ,medicine.drug - Abstract
Background: The genetic landscape of adult acute myeloid leukemias (AML) has been recently unravelled. This makes achievable the determination of a comprehensive profile of driver lesions for virtually all patients at diagnosis. Recent studies using multi-target minimal residual disease (MRD) strategies with around 1% sensitivity indicate that the clearance of all molecular events after chemotherapy is associated with better survival. To improve the clono-specificity and the sensitivity of this approach, after a precise determination of AML clonal composition, we combined cytogenetic, FISH, and high sensitivity deep sequencing technologies to monitor the MRD in a series of 69 patients. Methods: Forty-five consecutive patients reflecting the genetic diversity of AML were prospectively included and 24 patients were retrospectively studied. All patients received an anthracycline + cytarabine based regimen. The clonal architecture was established at diagnosis based on NGS-targeted resequencing (122 gene panel) and cytogenetic data. Lesions were next investigated in complete remission (CR). Based on the initial clonal composition, targeted resequencing panels were designed to improve the sensitivity by the use of unique molecular barcodes (Haloplex High Sensitivity, or HS-NGS assay). Cytogenetic events were evaluated by FISH. Results: In the 69 patients, a median of 4 genetic or chromosomal events were identified per patient (range 0-10). One patient had no evaluable target allowing MRD evaluation in 68/69 patients. To determine the threshold of detection of the HS NGS assay, we analyzed the frequency of mutant reads in multiple samples expected to be wild type for 31 given SNVs and 2 indels. A consensus threshold of detection was set at a variant allele frequency (VAF) of 0.2% for all lesions. In CR samples, early initiating events frequently persisted after treatment, especially mutations in DNMT3A, TET2, ASXL1, EZH2, IDH1, TP53, SRSF2, and U2AF1. Mutations in FLT3, NRAS, KIT, NPM1, CEBPA, WT1, IDH2 and BCOR were the most frequently cleared events. Seven patients did not reach CR after one course, and two had no available material after one course. In the 59 remaining patients, we tested whether the global response level of all targets was associated with prognosis. We used the median VAF of the first events of all clonal architectures to separate good responder from poor responders (i.e. VAF = 1.66%). At 2 years, there was a trend to lower leukemia free survival (LFS) probability in poor responders (31.7+/-9.9% vs 51.7+/-9.8%, p=0.08) with no translation in overall survival (OS). We next investigated if the persistence of two or more detectable markers was associated with prognosis. The 58 patients with more than one evaluable event were consequently separated in two groups: patients with 0 or 1 marker above the detection threshold after treatment (n=31), and patients with 2 or more detectable lesions (n=27). At 2 years, DFS was 64.9+/-9.3 % in patients with 0 or 1 detectable marker vs 19.8+/-8.7% in patients with 2 or more detectable markers (p=0.001). OS probability was higher in patients with 0 or 1 detectable marker 84+/-6.6% vs 57.1+/-10.5% (p=0.023). When focusing on the 40 patients with intermediate cytogenetics, persistence of 2 or more markers was associated with lower LFS (57+/-11.8% vs 19.4+/-10.5 p=0.0048) and with a trend to lower OS (85+/-8% vs 61+/-11.9% p=0.07). Similar results were observed when restricting the analyses to the 42 prospectively included patients (At 2 years: LFS 73+/-10% vs 24+/-10%, p=0.0026 and OS 90.2+/-6.6% vs 62.8+/-11.5%, p=0.036). In 50 patients with 3 or more identified events, the persistence of 3 or more markers after one course was associated with a very high risk of relapse (DFS 23.5+/-10.3 % vs 75.8 +/-7.5% at one year, p Conclusion Our study shows the high prognostic value of a personalized multi-target clono-specific MRD evaluation that can be used in nearly all AML patients. Detection of two or more events in more than 0.4% cells after one course is associated with lower survival, in particular in intermediate cytogenetic patients. Larger studies are needed to confirm the results and to evaluate if this strategy could be useful to guide treatment decisions. Disclosures No relevant conflicts of interest to declare.
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- 2016
15. New insights for pelvic radiation disease treatment: Multipotent stromal cell is a promise mainstay treatment for the restoration of abdominopelvic severe chronic damages induced by radiotherapy
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Luc Douay, Jan Voswinkel, Marc Benderitter, Sabine François, and Alain Chapel
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Oncology ,medicine.medical_specialty ,Pathology ,Histology ,Stromal cell ,business.industry ,medicine.medical_treatment ,Mesenchymal stem cell ,Cell Biology ,Frontier ,Cell therapy ,Clinical trial ,Radiation therapy ,Gastrointestinal disorder ,Internal medicine ,Genetics ,Medicine ,Stem cell ,business ,Adverse effect ,Molecular Biology ,Genetics (clinical) - Abstract
Radiotherapy may induce irreversible damage on healthy tissues surrounding the tumor. It has been reported that the majority of patients receiving pelvic radiation therapy show early or late tissue reactions of graded severity as radiotherapy affects not only the targeted tumor cells but also the surrounding healthy tissues. The late adverse effects of pelvic radiotherapy concern 5% to 10% of them, which could be life threatening. However, a clear medical consensus concerning the clinical management of such healthy tissue sequelae does not exist. Although no pharmacologic interventions have yet been proven to efficiently mitigate radiotherapy severe side effects, few preclinical researches show the potential of combined and sequential pharmacological treatments to prevent the onset of tissue damage. Our group has demonstrated in preclinical animal models that systemic mesenchymal stromal cell (MSC) injection is a promising approach for the medical management of gastrointestinal disorder after irradiation. We have shown that MSCs migrate to damaged tissues and restore gut functions after irradiation. We carefully studied side effects of stem cell injection for further application in patients. We have shown that clinical status of four patients suffering from severe pelvic side effects resulting from an over-dosage was improved following MSC injection in a compationnal situation.
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- 2013
16. In vitro and in vivo evidence for the long-term multilineage (myeloid, B, NK, and T) reconstitution capacity of ex vivo expanded human CD34+ cord blood cells
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Xiaxin Li, Monique Titeux, François Leteurtre, Marie-Catherine Giarratana, Luc Douay, Ladan Kobari, Laure Coulombel, Françoise Pflumio, and Brigitte Izac
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Cancer Research ,Myeloid ,T-Lymphocytes ,CD34 ,Antigens, CD34 ,Stem cell factor ,Mice, SCID ,Biology ,Mice ,Interleukin 21 ,Mice, Inbred NOD ,Granulocyte Colony-Stimulating Factor ,Genetics ,medicine ,Animals ,Humans ,Lymphocytes ,Lymphopoiesis ,Molecular Biology ,Cells, Cultured ,B-Lymphocytes ,Stem Cell Factor ,Graft Survival ,Hematopoietic Stem Cell Transplantation ,Membrane Proteins ,Cell Differentiation ,Cell Biology ,Hematology ,Fetal Blood ,Hematopoietic Stem Cells ,Hematopoiesis ,Killer Cells, Natural ,Haematopoiesis ,medicine.anatomical_structure ,Thrombopoietin ,Immunology ,Cancer research ,Bone marrow ,Stem cell ,Granulocytes - Abstract
The aim of the present report is to describe clinically relevant culture conditions that support the expansion of primitive hematopoietic progenitors/stem cells, with maintenance of their hematopoietic potential as assessed by in vitro assays and the NOD-SCID in vivo repopulating capacity.CD34(+) cord blood (CB) cells were cultured in serum-free medium containing stem cell factor, Flt3 ligand, megakaryocyte growth and development factor, and granulocyte colony-stimulating factor. After 14 days, the primitive functions of expanded and nonexpanded cells were determined in vitro using clonogenic cell (colony-forming cells, long-term culture initiating cell [LTC-IC], and extended [E]-LTC-IC) and lymphopoiesis assays (NK, B, and T) and in vivo by evaluating long-term engraftment of the bone marrow of NOD-SCID mice. The proliferative potential of these cells also was assessed by determining their telomere length and telomerase activity. Levels of expansion were up to 1,613-fold for total cells, 278-fold for colony-forming unit granulocyte-macrophage, 47-fold for LTC-IC, and 21-fold for E-LTC-IC. Lymphoid B-, NK, and T-progenitors could be detected. When the expanded populations were transplanted into NOD-SCID mice, they were able to generate myeloid progenitors and lymphoid cells for 5 months. These primitive progenitors engrafted the NOD-SCID bone marrow, which contained LTC-IC at the same frequency as that of control transplanted mice, with conservation of their clonogenic capacity. Moreover, human CD34(+)CDl9(-) cells sorted from the engrafted marrow were able to generate CD19(+) B-cells, CD56(+)CD3(-) NK cells, and CD4(+)CD8(+)alphabetaTCR(+) T-cells in specific cultures. Our expansion protocol also maintained the telomere length in CD34(+) cells, due to an 8.8-fold increase in telomerase activity over 2 weeks of culture. These experiments provide strong evidence that expanded CD34(+) CB cells retain their ability to support long-term hematopoiesis, as shown by their engraftment in the NOD-SCID model, and to undergo multilineage differentiation along all myeloid and the B-, NK, and T-lymphoid pathways. The expansion protocol described here appears to maintain the hematopoietic potential of CD34(+) CB cells, which suggests its relevance for clinical applications.
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- 2000
17. Importance of marrow dose on posttransplant outcome in acute leukemia
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L. Fouillard, Christine Perot, Jean-Pierre Jouet, Francis Bauters, Luc Douay, J. P. Laporte, Jacqueline Van Den Akker, Manuel Lopez, Norbert-Claude Gorin, Albert Najman, Myriam Labopin, Françoise Isnard, S Lesage, and Nassima Bellal
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Cancer Research ,medicine.medical_specialty ,medicine.medical_treatment ,Population ,Gastroenterology ,law.invention ,chemistry.chemical_compound ,Randomized controlled trial ,Mafosfamide ,law ,Internal medicine ,Acute lymphocytic leukemia ,Genetics ,medicine ,education ,Molecular Biology ,Acute leukemia ,education.field_of_study ,business.industry ,Cytogenetics ,Cell Biology ,Hematology ,medicine.disease ,Autotransplantation ,Surgery ,chemistry ,Stem cell ,business - Abstract
Several prospective randomized trials in acute myelocytic leukemia (AML) documented a lower relapse rate with autologous bone marrow transplantation (ABMT) than with conventional chemotherapy. However, they also identified some transplant difficulties, such as failure to collect sufficient numbers of stem cells, slow kinetics of engraftment, and a high transplant-related mortality that diminished or negated positive impact on overall survival. Data for ABMT are inconclusive in acute lymphocytic leukemia (ALL) in adults. We retrospectively analyzed patients with acute leukemia autografted with marrow purged with mafosfamide after January 1983 in our institution. The population comprised 229 consecutive patients; 165 with AML [123 in first remission (CR1), 32 in second remission (CR2)]; 61 with ALL (46 in CR1, 4 in CR2); and 3 with undifferentiated acute leukemia. All patients were autografted with marrow purged with mafosfamide. Mafosfamide was given at a constant dose of 50 μg/mL in 103 and adjusted individually to produce a CFU-GM LD 95 (5% residual CFU-GM post purging) in 126. The outcome was analyzed for correlation with patient characteristics, the disease including cytogenetics, and the graft itself. Prognostic factors identified by multivariate analysis were used to derive a prognostic classification. Patients receiving higher doses of marrow submitted to purging (>5.46 × 10 4 CFU-GM/kg) experienced a lower treatment-related mortality (RR = 0.11, p=0.005) and a higher leukemia-free (RR = 0.5, p=0.005) and overall survival (RR = 0.4, p=0.001). Patients receiving 5.46 × 10 4 CFU-GM/kg and doses actually infused post purging of ≤0.02 × 10 4 /kg had a treatment-related mortality of only 2 ± 2%, a leukemia-free survival of 70%, and an overall survival of 77 ± 7% at 10 years. In this study of autotransplantation for acute leukemia using mafosfamide-purged marrow, the stem cell dose used for purging and the intensity of purging were the most important factors predicting outcome.
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- 1999
18. Fifteen years of preclinical and clinical experiences about biotherapy treatment of lesions induced by accidental irradiation and radiotherapy
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Sabine François, Marc Benderitter, Luc Douay, Jan Voswinkel, and Alain Chapel
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medicine.medical_specialty ,Severe combined immunodeficiency ,Histology ,business.industry ,medicine.medical_treatment ,Mesenchymal stem cell ,Cell Biology ,Total body irradiation ,medicine.disease ,Frontier ,Surgery ,Radiation therapy ,Therapeutic approach ,Genetics ,medicine ,Radiodermatitis ,Stem cell ,business ,Adverse effect ,Molecular Biology ,Genetics (clinical) - Abstract
High dose radiation exposures involving medical treatments or accidental irradiation may lead to extended damage to the irradiated tissue. Alleviation or even eradication of irradiation induced adverse events is therefore crucial. Because developments in cell therapy have brought some hope for the treatment of tissues damages induced by irradiation, the Institute for Radiation and Nuclear Safety contributed to establish the clinical guidelines for the management of accidentally irradiated victims and to provide the best supportive care to patients all over the world. In the past 15 years, we contributed to develop and test cell therapy for protection against radiation side effects in several animal models, and we proposed mechanisms to explain the benefit brought by this new therapeutic approach. We established the proof of concept that mesenchymal stem cells (MSCs) migrate to damaged tissues in the nonobese diabetic/severe combined immunodeficiency immunotolerant mice model and in non-human primate after radiation exposure. We showed that the intravenous injection of MSCs sustains hematopoiesis after total body irradiation, improves wound healing after radiodermatitis and protects gut function from irradiation damages. Thanks to a tight collaboration with clinicians from several French hospitals, we report successful treatments of therapeutic/accidental radiation damages in several victims with MSC infusions for hematopoiesis correction, radio-induced burns, gastrointestinal disorders and protection homeostatic functions of gut management after radio-therapy.
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- 2013
19. In vitro generated Rh(null) red cells recapitulate the in vivo deficiency: a model for rare blood group phenotypes and erythroid membrane disorders
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Jean-Pierre Cartron, Anne Dubart-Kupperschmitt, Marie Cambot, Christelle Mazurier, Véronique Picard, Denis Clay, Julien Picot, Pierre Ripoche, Luc Douay, Nicolas Hebert, and Florence Canoui-Poitrine
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Reticulocytes ,Cellular differentiation ,Porphyria, Erythropoietic ,CD47 Antigen ,Anemia, Hemolytic, Congenital ,Cell Line ,Erythroid Cells ,Pregnancy ,Humans ,Progenitor cell ,RNA, Small Interfering ,Cells, Cultured ,Genetics ,Erythroid Precursor Cells ,Fetal Stem Cells ,Membrane Glycoproteins ,Rh-Hr Blood-Group System ,biology ,CD47 ,Genetic Diseases, Inborn ,Cell Differentiation ,Hematology ,Blood Proteins ,Fetal Blood ,Cell biology ,Adult Stem Cells ,Cell culture ,RHAG ,Membrane biogenesis ,biology.protein ,Female ,RNA Interference ,Anemia, Hypoplastic, Congenital ,Cell Adhesion Molecules ,Biogenesis ,Ex vivo - Abstract
Lentiviral modification combined with ex vivo erythroid differentiation was used to stably inhibit RhAG expression, a critical component of the Rh(rhesus) membrane complex defective in the Rh(null) syndrome. The cultured red cells generated recapitulate the major alterations of native Rh(null) cells regarding antigen expression, membrane deformability, and gas transport function, providing the proof of principle for their use as model of Rh(null) syndrome and to investigate Rh complex biogenesis in human primary erythroid cells. Using this model, we were able to reveal for the first time that RhAG extinction alone is sufficient to explain ICAM-4 and CD47 loss observed on native Rh(null) RBCs. Together with the effects of RhAG forced expression in Rh(null) progenitors, this strongly strengthens the hypothesis that RhAG is critical to Rh complex formation. The strategy is also promising for diagnosis purpose in order to overcome the supply from rare blood donors and is applicable to other erythroid defects and rare phenotypes, providing models to dissect membrane biogenesis of multicomplex proteins in erythroid cells, with potential clinical applications in transfusion medicine.
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- 2013
20. Caspase-3 Is Involved in the Signalling in Erythroid Differentiation by Targeting Late Progenitors
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Daniela Boehm, Christelle Mazurier, Dhouha Darghouth, Marie-Catherine Giarratana, Luc Douay, Laurence Harmand, Anne-Marie Faussat, HAL UPMC, Gestionnaire, Différenciation et prolifération des cellules souches adultes. application à la thérapie cellulaire hématopoiétique, Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche Saint-Antoine (UMRS893), Etablissement Français du Sang (EFS), EFS, Cytométrie et Imagerie Saint-Antoine (CISA), Unité Mixte de Service d'Imagerie et de Cytométrie (UMS LUMIC), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), CHU Trousseau [APHP], and Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)
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[SDV.MHEP.HEM] Life Sciences [q-bio]/Human health and pathology/Hematology ,Myeloid ,Cell division ,Cellular differentiation ,Red Cells ,lcsh:Medicine ,Apoptosis ,Biochemistry ,0302 clinical medicine ,hemic and lymphatic diseases ,Molecular Cell Biology ,lcsh:Science ,Erythroid Precursor Cells ,Cells, Cultured ,0303 health sciences ,Multidisciplinary ,Caspase 3 ,Stem Cells ,Cell Differentiation ,[SDV.MHEP.HEM]Life Sciences [q-bio]/Human health and pathology/Hematology ,Hematology ,Cell cycle ,Caspase Inhibitors ,Cell biology ,Enzymes ,Adult Stem Cells ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Medicine ,Cell Division ,Signal Transduction ,Research Article ,G2 Phase ,Biology ,Cell Growth ,03 medical and health sciences ,medicine ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Humans ,Cell Lineage ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Progenitor cell ,030304 developmental biology ,Cell Proliferation ,Cell growth ,lcsh:R ,Hematopoietic Stem Cells ,Hematopoiesis ,Enzyme Activation ,lcsh:Q ,Developmental Biology - Abstract
International audience; A role for caspase activation in erythroid differentiation has been established, yet its precise mode of action remains elusive. A drawback of all previous investigations on caspase activation in ex vivo erythroid differentiation is the lack of an in vitro model producing full enucleation of erythroid cells. Using a culture system which renders nearly 100% enucleated red cells from human CD34 + cells, we investigated the role of active caspase-3 in erythropoiesis. Profound effects of caspase-3 inhibition were found on erythroid cell growth and differentiation when inhibitors were added to CD34 + cells at the start of the culture and showed dose-response to the concentration of inhibitor employed. Enucleation was only reduced as a function of the reduced maturity of the culture and the increased cell death of mature cells while the majority of cells retained their ability to extrude their nuclei. Cell cycle analysis after caspase-3 inhibition showed caspase-3 to play a critical role in cell proliferation and highlighted a novel function of this protease in erythroid differentiation, i.e. its contribution to cell cycle regulation at the mitotic phase. While the effect of caspase-3 inhibitor treatment on CD34 + derived cells was not specific to the erythroid lineage, showing a similar reduction of cell expansion in myeloid cultures, the mechanism of action in both lineages appeared to be distinct with a strong induction of apoptosis causing the decreased yield of myeloid cells. Using a series of colony-forming assays we were able to pinpoint the stage at which cells were most sensitive to caspase-3 inhibition and found activated caspase-3 to play a signalling role in erythroid differentiation by targeting mature BFU-E and CFU-E but not early BFU-E.
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- 2013
21. A new congenital dysmegakaryopoietic thrombocytopenia (Paris-Trousseau) associated with giant platelet alpha-granules and chromosome 11 deletion at 11q23 [see comments]
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Rémi Favier, Josette Guichard, Luc Douay, D Cherif, Najet Debili, William Vainchenker, Roland Berger, and J Breton-Gorius
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Pathology ,medicine.medical_specialty ,biology ,Platelet disorder ,Immunology ,Paris-Trousseau syndrome ,GATA1 ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Chromosome aberration ,Von Willebrand factor ,biology.protein ,medicine ,Platelet ,Thrombopoiesis ,Jacobsen syndrome - Abstract
This study characterizes a new congenital thrombocytopenia with mild hemorrhagic tendency occurring in a woman and her child with the following features. We found a deletion of the distal part of one chromosome 11 [del(11)q23.3-->qter] that was detected by cytogenetic analysis and confirmed by chromosome painting in the two patients and also an increased number of bone marrow megakaryocytes (MKs), including numerous micromegakaryocytes (mMKs) associated with a normal platelet life span. A normal number of MK colonies in culture was observed with one third of them containing a few large MKs; however, these were always associated with mMKs identified by immunologic staining. A massive cell lysis was observed at the end of the maturation. Fifteen percent of the platelets in the peripheral blood showed giant alpha-granules resulting from the fusion of alpha-granules. These giant granules, which appeared in red on giemsa stain, had a mean diameter of 1.5 microns and showed all markers (detected at electron microscopy by immunogold method) of matrix and alpha-granule membrane, ie, von Willebrand factor, fibrinogen, CD41, CD62P (P-selectin); however, they differed from lysosomes because acid phosphatases were not present. These giant alpha-granules were unable to release their contents after stimulation by thrombin, in contrast to platelets with normal morphology. Abnormalities in bone marrow MK maturation that were detected at the electron microscopic level and that led to lysis of numerous MKs were responsible for thrombocytopenia and were similar in both patients. MK abnormalities are probably the consequence of the chromosome aberration. ETS 1 and FLI, two proto-oncogenes that appear to be essential with GATA1 for the normal expression of MK-specific genes, map to 11q23-q24 and are, thus, deleted in this thrombocytopenia. In conclusion, the association of all these abnormalities constitutes a new familial platelet disorder and may present a valuable model for exploring the role of some genes involved in the regulation of thrombopoiesis.
- Published
- 1995
22. Biological validation of bio-engineered red blood cell productions
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Marie-Catherine Giarratana, Luc Douay, Dhouha Darghouth, and Tiffany Marie
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Erythrocytes ,Reticulocytes ,Context (language use) ,CD47 Antigen ,Phosphatidylserines ,Biology ,Membrane Lipids ,Mice ,Reticulocyte ,Phagocytosis ,Erythrocyte Deformability ,medicine ,Animals ,Humans ,Erythropoiesis ,Leukapheresis ,Molecular Biology ,Cells, Cultured ,Fluorescent Dyes ,Macrophages ,Erythrocyte Membrane ,Cell Biology ,Hematology ,Erythrocyte Aging ,Fibroblasts ,Fluoresceins ,Hematopoietic Stem Cells ,Erythrophagocytosis ,In vitro ,Cell biology ,Red blood cell ,Haematopoiesis ,medicine.anatomical_structure ,Cell culture ,Immunology ,Molecular Medicine ,Stem cell ,Erythrocyte Transfusion - Abstract
The generation in vitro of cultured red blood cells (cRBC) could become an alternative to classical transfusion products. However, even when derived from healthy donors, the cRBC generated in vitro from hematopoietic stem cells may display alterations resulting from a poor controlled production process. In this context, we attempted to monitor the quality of the transfusion products arising from new biotechnologies. For that purpose, we developed an in vitro erythrophagocytosis (EP) test with the murine fibroblast cell line MS-5 and human macrophages (reference method). We evaluated 38 batches of cRBC, at the stage of reticulocyte, generated from CD34+ cells isolated from placental blood or by leukapheresis. We showed that (i) the EP test performed with the MS-5 cell line was sensitive and can replace human macrophages for the evaluation of cultured cells. (ii) The EP tests revealed disparities among the batches of cRBC. (iii) The viability of the cells (determined by calcein-AM test), the expression of CD47 (antiphagocytosis receptor) and the externalization of phosphatidylserine (PS, marker of phagocytosis) were not critical parameters for the validation of the cRBC. (iv) Conversely, the cell deformability determined by ektacytometry was inversely correlated with the intensity of the phagocytic index. Assuming that the culture conditions directly influence the quality of the cell products generated, optimization of the production mode could benefit from the erythrophagocytosis test.
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- 2012
23. Human induced pluripotent stem cells can reach complete terminal maturation: in vivo and in vitro evidence in the erythropoietic differentiation model
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Dominique Luton, Shaghayegh Rouzbeh, Laurent Kiger, Alain Chapel, Annelise Bennaceur-Griscelli, Noufissa Oudrhiri, Ladan Kobari, Wassim El-Nemer, Christelle Mazurier, Sabine François, Hélène Lapillonne, Alain Francina, Marie-Catherine Giarratana, Luc Douay, Nicolas Hebert, Frank Yates, Centre de Recherche Saint-Antoine (CR Saint-Antoine), Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Saint-Antoine [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Modèles de Cellules Souches Malignes et Thérapeutiques, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Sud - Paris 11 (UP11), Unité de Pathologie Moléculaire du Globule Rouge, Hospices Civils de Lyon (HCL)-Hôpital Edouard Herriot [CHU - HCL], Hospices Civils de Lyon (HCL), INSERM U473, Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Transfusion Sanguine, Paris, France, Inserm UMR_S 665, Paris, France, Université Paris Diderot, Sorbonne Paris Cité, UMR-S665, Paris, France, PRP-HOM/SRBE/LRTE, Institut de Radioprotection et de Sûreté Nucléaire (IRSN), Hôpital Beaujon [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), ATHENA, Irsn, Centre de Recherche Saint-Antoine (CRSA), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Université Paris-Sud - Paris 11 (UP11)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Laboratoire de Radiopathologie et de Thérapies Expérimentales (IRSN/PRP-HOM/SRBE/LRTE)
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KOSR ,Adult ,Erythrocytes ,[SDV]Life Sciences [q-bio] ,Cellular differentiation ,Induced Pluripotent Stem Cells ,Anemia, Sickle Cell ,Mice, SCID ,Biology ,In Vitro Techniques ,03 medical and health sciences ,Hemoglobins ,Mice ,0302 clinical medicine ,Mice, Inbred NOD ,Fetal hemoglobin ,Cell Adhesion ,Animals ,Humans ,Erythropoiesis ,Induced pluripotent stem cell ,Cells, Cultured ,030304 developmental biology ,0303 health sciences ,Induced stem cells ,Cell Differentiation ,Hematology ,Fibroblasts ,Amniotic Fluid ,Flow Cytometry ,3. Good health ,Cell biology ,[SDV] Life Sciences [q-bio] ,Endothelial stem cell ,Oxygen ,030220 oncology & carcinogenesis ,Female ,Stem cell ,Original Articles and Brief Reports ,Adult stem cell - Abstract
International audience; Background Human induced pluripotent stem cells offer perspectives for cell therapy and research models for diseases. We applied this approach to the normal and pathological erythroid differentiation model by establishing induced pluripotent stem cells from normal and homozygous sickle cell disease donors. Design and Methods We addressed the question as to whether these cells can reach complete erythroid terminal maturation notably with a complete switch from fetal to adult hemoglobin. Sickle cell disease induced pluripotent stem cells were differentiated in vitro into red blood cells and characterized for their terminal maturation in terms of hemoglobin content, oxygen transport capacity, deformability, sickling and adherence. Nucleated erythroblast populations generated from normal and pathological induced pluripotent stem cells were then injected into non-obese diabetic severe combined immunodeficiency mice to follow the in vivo hemoglobin maturation. Results We observed that in vitro erythroid differentiation results in predominance of fetal hemoglobin which rescues the functionality of red blood cells in the pathological model of sickle cell disease. We observed, in vivo, the switch from fetal to adult hemoglobin after infusion of nucleated erythroid precursors derived from either normal or pathological induced pluripotent stem cells into mice. Conclusions These results demonstrate that human induced pluripotent stem cells i) can achieve complete terminal erythroid maturation, in vitro in terms of nucleus expulsion and in vivo in terms of hemoglobin maturation; and ii) open the way to generation of functionally corrected red blood cells from sickle cell disease induced pluripotent stem cells, without any genetic modification or drug treatment. © 2012 Ferrata Storti Foundation.
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- 2012
24. Deep Proteomic Analysis of Human Erythropoiesis
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Frédérique Verdier, Patrick Mayeux, Virginie Salnot, Sarah Ducamp, Michael Dussiot, Catherine Lacombe, Emilie-Fleur Gautier, Marjorie Leduc, Marie-Catherine Giarratana, Yael Zermati, Luc Douay, and François Guillonneau
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Genetics ,Cell division ,Cell growth ,Immunology ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Cell biology ,Transcriptome ,Cell nucleus ,medicine.anatomical_structure ,Membrane protein ,Proteome ,medicine ,Erythropoiesis ,Progenitor cell - Abstract
*the first two authors are co-first authors Introduction. Erythropoiesis is a complex process starting from pluripotent medullary progenitors and leading to the production of highly specialized and enucleated erythrocytes. Two successive phases are generally distinguished: an amplification phase with intense proliferation of morphologically similar progenitors and a terminal differentiation phase with few cell divisions and strong cellular modifications. Although erythropoiesis is a continuous process, these modifications allow the identification of specific maturation stages and the passage from one stage to the following one seems to correlate with a cell division. Several transcriptomic analyses of erythroid differentiation have been published but only few and very limited proteomic studies have been reported. Since post transcriptomic modifications are responsible for a large part of the proteome variations, a direct proteomic analysis of the erythroid differentiation is required to accurately assess the modifications that occur during this process. Results. For this study, we used CD34+ cord blood progenitors and an optimized three step cell culture method allowing the production of highly synchronized cell populations of erythroid cells at various differentiation stages. Several cellular populations from erythroid progenitors up to reticulocytes were analyzed by a label-free analysis and mass spectrometry that led to the absolute quantification of more than 6000 proteins with a false discovery rate of less than 1% (n=3). Moreover, the relative expression of well-known stage-specific erythroid markers such as TFRC, BAND3 or GLUT1, transcription factors, heme biosynthesis enzymes followed the expected pattern. To complete this study, we performed a quantitative analysis of the repartition of proteins between the generated reticulocytes and the expelled nucleus (pyrenocyte). To do that, pyrenocytes and reticulocytes were sorted by FACS according to size, Hoechst 33342 and glycophorin A labelling. Equal numbers of reticulocytes and pyrenocytes were used to prepare peptides that were analyzed by mass spectrometry after iTRAQ labelling. These experiments allowed the quantitative repartition of 1153 proteins including most erythrocyte-specific membrane proteins. Conclusion. All these results significantly increase our knowledge of the protein expression pattern during erythropoiesis and should constitute a valuable data base for subsequent studies regarding both physiological and disordered erythropoiesis. Disclosures No relevant conflicts of interest to declare.
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- 2015
25. Proof of principle for transfusion of in vitro-generated red blood cells
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Sabine François, Laurent Kiger, Hélène Lapillonne, Germain Trugnan, Hélène Rouard, Séverine Jolly, Thierry Peyrard, Nicolas Hebert, Nathalie Mario, Tiffany Marie, Laurence Harmand, Christelle Mazurier, Marie-Catherine Giarratana, Luc Douay, Jean-Yves Devaux, Agnès Dumont, Pierre-Yves Le Pennec, Innocent Safeukui, Centre de Recherche Saint-Antoine (UMRS893), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Unité Mixte de Thérapie Cellulaire [Grenoble], CHU Grenoble-EFS, Immunologie moléculaire des parasites, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), CHU Saint-Antoine [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Institut de Radioprotection et de Sûreté Nucléaire (IRSN), Université Pierre et Marie Curie - Paris 6 (UPMC), Trafic Membranaire et Signalisation Dans les Cellules Epitheliales, Institut National de la Transfusion Sanguine [Paris] (INTS), Centre National de Référence pour les Groupes Sanguins (CNRGS), CNRGS, STMicroelectronics [Crolles] (ST-CROLLES), Service de médecine interne [Saint-Antoine], Université Pierre et Marie Curie - Paris 6 (UPMC)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Saint-Antoine [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU), Différenciation et prolifération des cellules souches adultes. application à la thérapie cellulaire hématopoiétique, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
Erythrocytes ,Plenary Paper ,Antigens, CD34 ,Mice, SCID ,Biochemistry ,Blood cell ,Hemoglobins ,Mice ,0302 clinical medicine ,Mice, Inbred NOD ,Erythropoiesis ,Cells, Cultured ,0303 health sciences ,education.field_of_study ,Hematology ,[CHIM.ORGA]Chemical Sciences/Organic chemistry ,Transfusion medicine ,Cell Differentiation ,Erythrocyte Aging ,Flow Cytometry ,3. Good health ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Blood Group Antigens ,Stem cell ,Erythrocyte Transfusion ,medicine.medical_specialty ,Cell Survival ,Immunology ,Population ,Transplantation, Heterologous ,Biology ,In Vitro Techniques ,03 medical and health sciences ,In vivo ,Internal medicine ,Erythrocyte Deformability ,medicine ,Animals ,Humans ,education ,030304 developmental biology ,Cell Proliferation ,Severe combined immunodeficiency ,Cell Biology ,medicine.disease ,Hematopoietic Stem Cells ,Red blood cell - Abstract
In vitro RBC production from stem cells could represent an alternative to classic transfusion products. Until now the clinical feasibility of this concept has not been demonstrated. We addressed the question of the capacity of cultured RBCs (cRBCs) to survive in humans. By using a culture protocol permitting erythroid differentiation from peripheral CD34+ HSC, we generated a homogeneous population of cRBC functional in terms of their deformability, enzyme content, capacity of their hemoglobin to fix/release oxygen, and expression of blood group antigens. We then demonstrated in the nonobese diabetes/severe combined immunodeficiency mouse that cRBC encountered in vivo the conditions necessary for their complete maturation. These data provided the rationale for injecting into one human a homogeneous sample of 1010 cRBCs generated under good manufacturing practice conditions and labeled with 51Cr. The level of these cells in the circulation 26 days after injection was between 41% and 63%, which compares favorably with the reported half-life of 28 ± 2 days for native RBCs. Their survival in vivo testifies globally to their quality and functionality. These data establish the proof of principle for transfusion of in vitro–generated RBCs and path the way toward new developments in transfusion medicine. This study is registered at http://www.clinicaltrials.gov as NCT0929266.
- Published
- 2011
26. Red blood cells from induced pluripotent stem cells: hurdles and developments
- Author
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Luc Douay, Hélène Lapillonne, and Christelle Mazurier
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Pluripotent Stem Cells ,medicine.medical_specialty ,Hematology ,Erythrocytes ,Cell growth ,Context (language use) ,Biology ,Regenerative medicine ,Cell biology ,Blood cell ,Red blood cell ,Mice ,medicine.anatomical_structure ,Internal medicine ,Immunology ,medicine ,Animals ,Humans ,Blood Transfusion ,Stem cell ,Induced pluripotent stem cell - Abstract
In the context of chronic blood supply difficulties, generating cultured red blood cells (cRBCs) in vitro after amplification of stem cells makes sense. This review will focus on the recent findings about the generation of erythroid cells from induced pluripotent stem (iPS) cells and deals with the hurdles and next developments that will occur.The most proliferative source of stem cells for generating cRBCs is the cord blood, but this source is limited in terms of hematopoietic stem cells and dependent on donations. Pluripotent stem cells are thus the best candidates and potential sources of cRBCs. Critical advances have led towards the in-vitro production of functional RBCs from iPS cells in the last few years.Because iPS cells can proliferate indefinitely and can be selected for a phenotype of interest, they are potential candidates to organize complementary sources of RBCs for transfusion. Proof of concept of generating cRBCs from iPS cells has been performed, but the procedures need to be optimized to lead to clinical application in blood transfusion. Several crucial points remain to be resolved. Notably these include the choice of the initial cell type to generate iPS cells, the method of reprogramming, that is, to ensure the safety of iPS cells as clinical grade, the optimization of erythrocyte differentiation, and the definition of good manufacturing practice (GMP) conditions for industrial production.
- Published
- 2011
27. Human Fetal Liver: An In Vitro Model of Erythropoiesis
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Guillaume Pourcher, Marie-Catherine Giarratana, Luc Douay, Hélène Lapillonne, Christelle Mazurier, Ladan Kobari, Daniela Boehm, and Yé Yong King
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KOSR ,lcsh:Internal medicine ,Article Subject ,CD34 ,Cell Biology ,Biology ,Embryonic stem cell ,Cell biology ,Endothelial stem cell ,Haematopoiesis ,Immunology ,Stem cell ,Induced pluripotent stem cell ,lcsh:RC31-1245 ,Molecular Biology ,Adult stem cell ,Research Article - Abstract
We previously described the large-scale production of RBCs from hematopoietic stem cells (HSCs) of diverse sources. Our present efforts are focused to produce RBCs thanks to an unlimited source of stem cells. Human embryonic stem (ES) cells or induced pluripotent stem cell (iPS) are the natural candidates. Even if the proof of RBCs production from these sources has been done, their amplification ability is to date not sufficient for a transfusion application. In this work, our protocol of RBC production was applied to HSC isolated from fetal liver (FL) as an intermediate source between embryonic and adult stem cells. We studied the erythroid potential of FL-derived CD34+cells. In thisin vitromodel, maturation that is enucleation reaches a lower level compared to adult sources as observed for embryonic or iP, but, interestingly, they (i) displayed a dramaticin vitroexpansion (100-fold more when compared to CB CD34+) and (ii) 100% cloning efficiency in hematopoietic progenitor assays after 3 days of erythroid induction, as compared to 10–15% cloning efficiency for adult CD34+cells. This work supports the idea that FL remains a model of study and is not a candidate forex vivoRBCS production for blood transfusion as a direct source of stem cells but could be helpful to understand and enhance proliferation abilities for primitive cells such as ES cells or iPS.
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- 2011
28. Red blood cell generation from human induced pluripotent stem cells: perspectives for transfusion medicine
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Hélène Puccio, G. Andreu, Laurent Kiger, Philippe Tropel, Stéphane Viville, Isabelle Zanella-Cléon, Marie Wattenhofer-Donzé, Christelle Mazurier, Nicolas Hebert, Marie-Catherine Giarratana, Luc Douay, Ladan Kobari, Alain Francina, and Hélène Lapillonne
- Subjects
KOSR ,Erythrocytes ,Induced Pluripotent Stem Cells ,Cell Culture Techniques ,Editorials and Perspectives ,Embryoid body ,Biology ,Cell Line ,03 medical and health sciences ,0302 clinical medicine ,Humans ,Induced pluripotent stem cell ,030304 developmental biology ,0303 health sciences ,Induced stem cells ,Cell Differentiation ,Hematology ,Embryonic stem cell ,3. Good health ,Cell biology ,030220 oncology & carcinogenesis ,Immunology ,Cytokines ,Original Article ,Stem cell ,Erythrocyte Transfusion ,Adult stem cell ,Human embryonic stem cell line - Abstract
Background Ex vivo manufacture of red blood cells from stem cells is a potential means to ensure an adequate and safe supply of blood cell products. Advances in somatic cell reprogramming of human induced pluripotent stem cells have opened the door to generating specific cells for cell therapy. Human induced pluripotent stem cells represent a potentially unlimited source of stem cells for erythroid generation for transfusion medicine.Design and Methods We characterized the erythroid differentiation and maturation of human induced pluripotent stem cell lines obtained from human fetal (IMR90) and adult fibroblasts (FD-136) compared to those of a human embryonic stem cell line (H1). Our protocol comprises two steps: (i) differentiation of human induced pluripotent stem cells by formation of embryoid bodies with indispensable conditioning in the presence of cytokines and human plasma to obtain early erythroid commitment, and (ii) differentiation/maturation to the stage of cultured red blood cells in the presence of cytokines. The protocol dispenses with major constraints such as an obligatory passage through a hematopoietic progenitor, co-culture on a cellular stroma and use of proteins of animal origin.Results We report for the first time the complete differentiation of human induced pluripotent stem cells into definitive erythrocytes capable of maturation up to enucleated red blood cells containing fetal hemoglobin in a functional tetrameric form.Conclusions Red blood cells generated from human induced pluripotent stem cells pave the way for future development of allogeneic transfusion products. This could be done by banking a very limited number of red cell phenotype combinations enabling the safe transfusion of a great number of immunized patients.
- Published
- 2010
29. Consensus guidance for banking and supply of human embryonic stem cell lines for research purposes
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Peter W. Andrews, Catriona Crombie, Raimund Strehl, Hung-Chih Kuo, Tsuneo Takahashi, Jonathan M. Auerbach, Rosemarie Kirzner, Tenneille Ludwig, Glyn Stacey, Andre Bh Choo, Rodney Turner, Jyotsna Dhawan, Jennifer Moody, Fanyi Zeng, Neta Lavon, Shelly Tannenbaum, Joseph Itskovitz-Eldor, Dong-Wook Kim, Sun Kyung Oh, Derek J. Hei, Patricia Olson, Hye-Yeong Ha, Alan Colman, Wannshin Chen, Timothy Dyke, Lars Ährlund-Richter, Maneesha S. Inamdar, Lodovica Borghese, D.M. Collins, Javard Arias-Diaz, Heli Scotman, Nissim Benvenisty, Mary Laughlin, Benjamin Reubinoff, Christine L. Mummery, Paul A. De Sousa, Sorapop Kiatpongsan, Yukio Nakamura, K. Bruce, Steve Oh, Duncan Baker, Andras Nagy, Augustin Zapata, Allan J. Robins, Qi Zhou, Majlinda Lakov, Shin-Ichi Nishikawa, Frida Holm, Stefanie Terstegge, Jeremy M. Crook, Gyan Mishra, Peter Zandstra, Maria Mileikovskaia, Robin Buckle, Barbara B. Knowles, Pablo Menendez, Heather M. Rooke, Oliver Brüstle, Ray Cypess, John Yu, Dong-Ryul Lee, Manuel Alvarez, Rebecca Skinner, Outi Hovatta, Timo Tuuri, Sonia Stefanovic, Michel Puceat, Jon K. Sherlock, John Macauley, Paul Gokhale, Sohel Talib, Shiaw-Min Hwang, Lena Eriksson, Meri T. Firpo, Luc Douay, Nancy Jessie, Carlos Simón, Anna E. Michalska, Timo Otonkoski, David Smith, Lyn Healy, Xuetao Pei, Phillipe Menasche, Aleš Hampl, Michele Greene, Clive Morris, Victor Rumayor, Joeri Borstlap, Hyun-Sook Park, Karen Dyer Montgomery, Charles J. Hunt, Milind Patole, Douglas Sipp, Kristiina Rajala, Guillaume Blin, Ronald D.G. McKay, Martin F. Pera, Clive Glover, Rrobert Taft, Carine Camby, Rosario Isasi, Dalit Ben-Josef, Petr Dvořák, Claire Fitzgerald, Stephen L. Minger, and Norio Nakatsuji
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Cancer Research ,Scientific progress ,Cell Biology ,Bioethics ,Embryonic stem cell ,Variety (cybernetics) ,Cell Line ,Embryo Research ,Humans ,Stem cell line ,Engineering ethics ,Business ,Stem cell ,Induced pluripotent stem cell ,Embryonic Stem Cells ,Biological Specimen Banks - Abstract
The work on human embryonic stem cells is being performed with a variety of cell lines using a variety of culture conditions; a situation that makes standardisation between projects and publications very difficult. Clearly the consequence of using such cells would be wasted time and resources but, more seriously, the generation of erroneous data in the literature which could both confuse and delay scientific progress in this area. This guidance document represents the outcome of the first meeting of the group named The International Stem Cell Banking Initiative held in October 2007. The document has been prepared from the perspective of hESC culture but, in many respects, is broadly applicable to all human stem cell lines including induced pluripotent stem cell lines.
- Published
- 2009
30. Ex Vivo Generation of Human Red Blood Cells: A New Advance in Stem Cell Engineering
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Marie-Catherine Giarratana and Luc Douay
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Haematopoiesis ,Stromal cell ,medicine.anatomical_structure ,Chemistry ,Cord blood ,medicine ,CD34 ,Erythropoiesis ,Bone marrow ,Stem cell ,Ex vivo ,Cell biology - Abstract
We describe a technological approach permitting the massive expansion of CD34(+) stem cells and their 100% conversion ex vivo into mature red blood cells (RBC). The protocol comprises three steps: a first step consisting of cell proliferation and induction of erythroid differentiation in a liquid medium without serum in the presence of growth factors (GF), a second based on a model reconstitution of the medullar microenvironment (ME) (human MSC or murine stromal cells) in the presence of GF, and a third in the presence of the ME alone, without any GF. This work highlights the impact of the ex vivo microenvironment on the terminal maturation of erythroid cells. A critical point is that the RBC generated in vitro have all the characteristics of functional native adult RBC. Moreover, this new concept of 'cultured RBC' (cRBC) is important for basic research into terminal erythropoiesis and has major clinical implications, especially in transfusion medicine. The three-step protocol can be adapted to use hematopoietic stem cells (HSC) from diverse sources: peripheral blood, bone marrow or cord blood.
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- 2009
31. Cultured Red Blood Cells: A New Advance in Stem Cell Engineering
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Luc Douay
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business.industry ,Hemostasis ,Immunology ,Medicine ,Hemoglobin ,Stem cell ,business ,Cell biology - Abstract
For several years, researchers have been trying to find a substitute for red blood cells (RBC). The development of chemical or natural molecules to replace hemoglobin has nevertheless proved difficul
- Published
- 2008
32. Naive and GMP-expanded CD34+ cells (ProtheraCytes®) have the same characteristics and potential capacities for heart regeneration after acute myocardial infarct
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Claire Saucourt, Brigitte Birebent, Anthony Criquet, Hélène Rouard, Philippe Henon, Valat Christophe, Luc Douay, Laurence Harmand, Amandine Merlin, and Sandrine Vogt
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Cancer Research ,Transplantation ,medicine.medical_specialty ,business.industry ,Cd34 cells ,Regeneration (biology) ,Immunology ,Cell Biology ,medicine.disease ,Oncology ,Internal medicine ,Cardiology ,Immunology and Allergy ,Medicine ,Myocardial infarction ,business ,Genetics (clinical) - Published
- 2015
33. Clonal Architecture of Relapsed MLL-AF9 Acute Myeloid Leukemia in a Child
- Author
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François Delhommeau, Ruoping Tang, Fanny Fava, Chrystele Bilhou-Nabera, Luc Douay, Hélène Lapillonne, Pierre Hirsch, Hélène Boutroux, and Guy Leverger
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Genetics ,Myeloid ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Trisomy 8 ,Biochemistry ,Minimal residual disease ,Somatic evolution in cancer ,Germline ,Frameshift mutation ,Leukemia ,medicine.anatomical_structure ,medicine ,Cancer research - Abstract
Introduction Acute myeloid leukemia (AML) is an aggressive malignancy caused by the accumulation of multiple oncogenetic mutations occurring in a single lineage of hematopoietic progenitors. AML is rare in children and the mutations found are partially different from those in adults, and for some with a lower frequency. Thus, clonal evolution leading to pediatric AML may be specific, and has not been described yet. Methods To define clonal evolution from diagnosis to relapse, we performed whole exome sequencing in matched trio of specimens (diagnosis, germline and relapse) in a 9-years old girl presenting AML FAB M5a with t(9;11)(p22;q23) MLL-AF9 and trisomy 8. At diagnosis, we focused on 3 non-silent somatic mutations candidate for leukemogenesis process, confirmed by Sanger method: EED (R355*), GSDMC (R40*) and ELK1 (3’ UTR). In the same time, we performed cell cultures from bone marrow mononucleated cells at diagnosis. CD34 and CD38 cells were cultured either in liquid long term culture medium (LTC IC) or methylcellulose medium. Results: A total of 512 colonies were collecte. Our 3 interest mutations and trisomy 8 were tracked by allele-specific PCR, and MLL rearrangement detected by FISH, individually in 267 from the 512 colonies. Exploitable results were found in 164 colonies. Through these results in the different cell populations, we were able to establish the clonal architecture at diagnosis. MLL-AF9 fusion and EED mutation were found together as the first concomitant occurring events in the leukemic clone. Then genotyping of the colonies demonstrated that ELK1 mutation, GSDMC mutation, and trisomy 8 were successively acquired. Additional later mutations such as ASXL1 (frameshift), PTPN11 (E76K), EMP2 (3’UTR) and DGCR14 (P314S) were detected in the relapse sample. Discussion The 3 mutations studied in the colonies may impact the progression of the leukemic clone by dysregulating several cellular pathways and networks. First, EED is an essential non-catalytic subunit of the polycomb repressive complex 2 (PRC2) which mediates gene silencing through catalysis of histone H3K27 methylation. PRC2 is known to be enhanced in solid neoplasms such as prostate cancer. On the contrary, in myeloid malignancies and myelodysplasic syndromes, it has been recently demonstrated that mutations involving PRC2 subunits (EED, SUZ12 and EZH1/2) were hypomorphic. These loss-of-functions mutations were responsible for chromatin relaxation and induced transcription of genes promoting self-renewal such as HOXA9. Nevertheless, recent sh-RNA studies in a murine model of MLL-AF9 leukemia demonstrated that residual PRC2 enzymatic activity after EED mutation is needed to unable leukemia growth. These data are coherent with our finding that EED mutation is an early event in leukemogenesis, in cooperation with MLL-AF9 rearrangement. Secondly, ELK1 is targeted by RAS-MAPK pathway, thus its mutation can confer an increased proliferation potential when acquired by the leukemic clone, after its maturation has been blocked and its self-renewal increased through previous MLL rearrangement and EED mutation. Finally, GSDMC may be implicated in monocyte count regulation, and mutated in other neoplasms such as melanoma. As a consequence, it is likely that its mutation occurs lately in the evolution of the monoblastic leukemic clone of our patient. The latest event in the clonal evolution in our patient at diagnosis is the acquisition of trisomy 8. Conclusion This study highlights the clonal evolution in one pediatric AML, and paves the way for further studies to better understand clonal evolution in children. Elucidating, the succession and the cooperation between driver and secondary mutations, is important for both understanding leukemogenesis and developing innovative therapeutic agents targeting founding anomalies in the leukemic clone at its most precocious stage. Moreover, discovering clonal architecture also unable to find new minimal residual disease markers to assess the therapeutic response and risk stratification. Disclosures No relevant conflicts of interest to declare.
- Published
- 2014
34. Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo
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Thi My Anh Neildez-Nguyen, Dominique Thierry, Morad Bensidhoum, Marie-Catherine Giarratana, Luc Douay, Michael C. Marden, Vincent Moncollin, Ladan Kobari, and Henri Wajcman
- Subjects
Erythrocytes ,Time Factors ,Cellular differentiation ,Biomedical Engineering ,CD34 ,Bioengineering ,Antigens, CD34 ,Cell Separation ,Mice, SCID ,Biology ,Applied Microbiology and Biotechnology ,Culture Media, Serum-Free ,Hemoglobins ,Mice ,medicine ,Animals ,Humans ,Progenitor cell ,Cells, Cultured ,Stem Cells ,Cell Differentiation ,Fetal Blood ,Flow Cytometry ,Hematopoietic Stem Cells ,Cell biology ,Endothelial stem cell ,Perfusion ,Haematopoiesis ,Red blood cell ,Kinetics ,medicine.anatomical_structure ,Cord blood ,Immunology ,Molecular Medicine ,Stem cell ,Cell Division ,Biotechnology - Abstract
New sources of red blood cells (RBCs) would improve the transfusion capacity of blood centers. Our objective was to generate cells for transfusion by inducing a massive proliferation of hematopoietic stem and progenitor cells, followed by terminal erythroid differentiation. We describe here a procedure for amplifying hematopoietic stem cells (HSCs) from human cord blood (CB) by the sequential application of specific combinations of growth factors in a serum-free culture medium. The procedure allowed the ex vivo expansion of CD34+ progenitor and stem cells into a pure erythroid precursor population. When injected into nonobese diabetic, severe combined immunodeficient (NOD/SCID) mice, the erythroid cells were capable of proliferation and terminal differentiation into mature enucleated RBCs. The approach may eventually be useful in clinical transfusion applications.
- Published
- 2002
35. Long-term quantitative bio-distribution and side effects of human mesenchymal stem cells (hMSCs) engraftment in NOD/SCID mice following irradiation
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Marc Benderitter, Alain Chapel, Sabine François, Luc Douay, and B. Usunier
- Subjects
Cancer Research ,Transplantation ,Chemistry ,Immunology ,Mesenchymal stem cell ,Cell Biology ,Nod ,Scid mice ,Oncology ,Cancer research ,Immunology and Allergy ,Irradiation ,Bio distribution ,Genetics (clinical) - Published
- 2014
36. Mesenchymal stem cells transplants after pelvic radiotherapy limits the development of radiation-induced fibrosis, without promoting the residual tumor growth
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Alain Chapel, Bruno l’Homme, Marc Benderitter, Luc Douay, and Sabine François
- Subjects
Cancer Research ,Transplantation ,Pathology ,medicine.medical_specialty ,business.industry ,Radiation induced fibrosis ,medicine.medical_treatment ,Immunology ,Mesenchymal stem cell ,Radiation induced ,Cell Biology ,medicine.disease ,Radiation therapy ,medicine.anatomical_structure ,Oncology ,Prostate ,Fibrosis ,medicine ,Immunology and Allergy ,Tumor growth ,business ,Pelvic radiotherapy ,Genetics (clinical) - Published
- 2014
37. Experimental culture conditions are critical for ex vivo expansion of hematopoietic cells
- Author
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Luc Douay
- Subjects
Immunology ,Population ,CD34 ,Cell Culture Techniques ,Bone Marrow Cells ,Cell Separation ,Biology ,Culture Media, Serum-Free ,medicine ,Animals ,Humans ,Progenitor cell ,education ,Cells, Cultured ,education.field_of_study ,Blood Cells ,Bone Marrow Purging ,Graft Survival ,Hematopoietic Stem Cell Transplantation ,Reproducibility of Results ,Hematology ,Fetal Blood ,Hematopoietic Stem Cells ,Bone marrow purging ,Cell biology ,Culture Media ,Haematopoiesis ,medicine.anatomical_structure ,Cord blood ,Receptors, Complement 3b ,Cattle ,Bone marrow ,Stem cell ,Cell Division - Abstract
The ex vivo expansion of hematopoietic stem cells (HSC) for clinical use is now recognized to be a feasible and very promising approach for hematotherapy. Expansion of specific HSC subsets is required for different clinical applications, for example, to increase the number of mature cells, to produce specific cells for adoptive therapy, or to increase the number of primitive stem cells available for engraftment. Although hematopoietic growth factors can play an important role in this setting, in this review we emphasize that other variables affect the outcome of stem and progenitor cell expansion. These variables include the serum supplement, the purity of CD34(+) cells, the initial cell concentration, and the duration of culture. It is also essential to define standard culture conditions for normal stem cells and to limit or prevent expansion of residual tumor cells. In clinical applications, determination of the hematopoietic value of the expanded population is mandatory. Thus, we have to demonstrate the expansion of primitive hematopoietic progenitor and stem cells, with maintenance of their hematopoietic potential as assessed by in vitro or in vivo assays. We draw attention to the challenges in the clinical application of ex vivo expansion. These include the establishment of well-defined experimental conditions and the determination of the hematopoietic value of the expanded grafts, whatever the graft source: bone marrow, mobilized peripheral blood, or cord blood. Future studies hopefully will optimize these procedures and allow not only expansion but engineering of defined cellular functions as HSCs grow under defined conditions.
- Published
- 2001
38. The Cultured Red Blood Cell: A Study Tool with Therapeutic Perspectives
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Marie-Catherine Giarratana and Luc Douay
- Subjects
Blood transfusion ,medicine.medical_treatment ,Cell Biology ,Cell lineage ,Biology ,Cell therapy ,Haematopoiesis ,Red blood cell ,medicine.anatomical_structure ,Cell transplantation ,Antigen ,Immunology ,medicine ,Ex vivo expansion ,Molecular Biology ,Developmental Biology - Published
- 2005
39. In Vitro Production of Erythrocytes
- Author
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Luc Douay
- Subjects
medicine.medical_specialty ,Immunology ,Transfusion medicine ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Embryonic stem cell ,Haematopoiesis ,medicine.anatomical_structure ,Cord blood ,Erythrocyte differentiation ,medicine ,Bone marrow ,Stem cell ,Induced pluripotent stem cell - Abstract
Abstract SCI-39 The generation of red blood cells (RBCs) in vitro using biotechnologies could represent an interesting alternative to classical transfusion products, in that it would combine adequate supplies with the specific production of blood products of a particular phenotype and the reduction of infection risks. This presentation will review how it is now possible to obtain in vitro complete maturation of the erythroid line to the stage of enucleation, starting from hematopoietic stem cells (HSCs) from peripheral blood, bone marrow or umbilical cord blood, or embryonic stem cells or adult pluripotent stem cells (induced pluripotent stem cells, iPSCs). This presentation will discuss how the functionality of cultured human RBCs (cRBCs) is settled in terms of deformability, hemoglobin maturation, oxygen carrying capacity, enzyme content, and terminal maturation from the reticulocyte stage to mature RBC after infusion into the NOD/SCID mouse model. The clinical feasibility of this concept has recently been demonstrated by reporting that cRBCs generated in vitro from peripheral HSCs under GMP conditions encounter in vivo the conditions required for their maturation and that they persist in the circulation for several weeks in humans. These data have established the proof of principle for transfusion of in vitro-generated RBCs and the pathway toward new developments in transfusion medicine. The most proliferative source of stem cells for generating cRBCs is cord blood, but it is limited in terms of HSCs and is dependent on donations. Pluripotent stem cell technology represents a potentially unlimited source of RBCs and opens the door to the development of a new generation of allogeneic transfusion products. Because iPSCs can be selected for a phenotype of interest, they are obviously the best candidate for organizing complementary sources of RBCs for transfusion. It is established that only three human iPSC clones would have been sufficient to match more than 99 percent of the patients in need of RBC transfusions. As a whole, a very limited number of RBC clones would provide for the needs of most alloimmunized patients and those with a rare blood group. Generating cRBCs from iPSCs has been done but needs to be optimized to lead to a clinical application in blood transfusion. Several crucial points remain to be resolved, notably, the choice of the initial cell type, the method of reprogramming (i.e., to ensure the safety of the iPSCs and to ensure their clinical grade), the optimization of the erythrocyte differentiation, and the definition of GMP conditions for industrial production. Assuming that in vitro large-scale cultured RBC production efficiently operates in the near future, this presentation will highlight the potential applications for alloimmunized patients and those with a rare blood group. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2012
40. One hundred twenty-five adult patients with primary acute leukemia autografted with marrow purged by mafosfamide: a 10-year single institution experience
- Author
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L. Fouillard, Jean-Pierre Jouet, Luc Douay, Myriam Labopin, Françoise Isnard, M.P. Noel-Walter, Marc Lopez, Jean-Philippe Laporte, Stachowiak J, and Lesage S
- Subjects
Adult ,Male ,medicine.medical_specialty ,Cyclophosphamide ,Adolescent ,Immunology ,Biochemistry ,Gastroenterology ,Transplantation, Autologous ,chemistry.chemical_compound ,Mafosfamide ,Internal medicine ,Antineoplastic Combined Chemotherapy Protocols ,Medicine ,Humans ,Acute leukemia ,Leukemia ,business.industry ,Bone Marrow Purging ,Graft Survival ,Remission Induction ,Cell Biology ,Hematology ,Total body irradiation ,Middle Aged ,medicine.disease ,Prognosis ,Combined Modality Therapy ,Surgery ,Transplantation ,Survival Rate ,Regimen ,medicine.anatomical_structure ,Treatment Outcome ,chemistry ,Acute Disease ,Multivariate Analysis ,Female ,Bone marrow ,business ,medicine.drug ,Follow-Up Studies - Abstract
A total of 125 acute leukemia adult patients were autografted with bone marrow (BM) purged by mafosfamide (ASTA Z) during the period of January 1983 to January 1993. The median follow-up period was 64 months (range, 3 to 126). There were 84 acute myeloblastic leukemias (AMLs) and 41 acute lymphoblastic leukemias (ALLs). At time of autologous BM transplantation (ABMT); 64 AMLs were in first complete remission (CR1), and 20 were in second CR (CR2); 35 ALL were in CR1, and 6 were in CR2. The median age of the patients was 33 years (range, 16 to 55). The median interval between achieving CR and autografting was 5 months (range, 1.3 to 23). The pretransplant regimen consisted of cyclophosphamide (120 mg/kg) and total body irradiation. All patients were grafted with autologous BM treated in vitro with mafosfamide used at levels individually adjusted in 95 patients and at a standard dose in 30 patients. The initial richness in granulomacrophagic progenitors (CFU-GM) of the harvested BMs was 5.16 x 10(4) CFU-GM/kg (range, 0.55 to 33). After mafosfamide purging, the residual CFU-GM number was 0.021 x 10(4)/kg (range, 0 to 1.78). The probability of successful engraftment was significantly higher and the time to engraftment was significantly shorter in ALL. Of 33 patients grafted with BM containing no residual CFU-GM, those with AML (n = 22) had platelet recoveries that were significantly longer than those for AML patients receiving BM with residual CFU-GM. At 8 years, patients autografted in CR1 for AML and ALL had a leukemia-free survival (LFS) of 58% and 56%, respectively, with a relapse incidence (RI) of 25% and 37%, respectively. Patients autografted in CR2 for AML had an LFS of 34% and an RI of 48% at 5 years. The incidence of late relapses was significantly higher in ALLs. By multivariate analysis, four factors were found to influence favorably engraftment in addition to a diagnosis of ALL, a younger age, ABMT performed in CR1, the adjusted dose technique of purging, and a shorter interval from CR to ABMT. Two factors were correlated with a better outcome. (1) The LFS was significantly higher and the transplant-related mortality significantly lower in patients who received richer BM. (2) The RI was significantly lower in patients autografted within 150 days from CR. Our results reinforce the view that ABMT is one approach to improve the outcome of adult patients with acute leukemia.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1994
41. Involvement of STAT3 Transcription Factor in Disseminated Nasal-Type Natural Killer Cell Lymphomas
- Author
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Paul Coppo, Norbert Claude Gorin, Félix Agbalika, Philippe Gaulard, Valérie Gouilleux-Gruart, Peggy Dartigues, Sandrine Bouchet, Luc Douay, Yenlin Huang, and Kaiss Lassoued
- Subjects
Lymphokine-activated killer cell ,biology ,CD3 ,Immunology ,Cell ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Lymphoma ,Natural killer cell ,Interleukin 21 ,medicine.anatomical_structure ,Cell culture ,Cancer research ,medicine ,biology.protein ,Cytotoxic T cell - Abstract
Disseminated nasal-type natural killer (NK) cell lymphoma is an aggressive disease with very poor prognosis. The usual chemoresistance of this disease led us to explore the possible role of the transcription factor STAT3 in oncogenesis. For this, we established and characterized a continuous interleukin (IL)2-dependent NK cell line (MEC04) from a patient with a fatal nasal-type NK cell lymphoma. MEC04 cells phenotype was CD3−, CD7+, TCR−, CD16−, CD56+bright, p58−, p70−, NKP−, and NKG2A+, they harbored poor cytotoxic activity against K562 cells, and spontaneously secreted interferon-γ, IL-10 and vascular-endothelium growth factor in vitro, suggesting that malignant cells arise from the CD56+bright regulatory NK cell population. Injection of MEC04 cells to NOD/SCID mice led to a fatal multiorgan infiltration by malignant cells. We show by immunohistochemistry that STAT3 is phosphorylated in Y705 dimerization residue in MEC04 and YT cell lines, and on biopsies from 6/6 patients with nasal-type NK cell lymphoma, suggesting that STAT3 may be oncogenic in this disease. By contrast, Y705 residue was not phosphorylated on 2 patients with hepatosplenic γδT-cell lymphoma. By confocal microscopy, we showed that STAT3 was located in nucleus in MEC04 cells. Y705 STAT3 phosphorylation involved JAK2, since exposure of cells to AG490 inhibitor inhibited Y705 STAT3 phosphorylation, and correlated in a dose-dependent growth inhibition at 24 hours and 48 hours. By using transducible TAT-STAT3b or TAT-STAT3Y705F recombinant proteins (a dominant-negative form of STAT3 [STAT3b isoform], and a STAT3 protein mutated on Y705 residue which prevents STAT3 dimerization [STAT3Y705F]), we were able to demonstrate that inhibition of STAT3 activation increased mortality and decreased proliferation by more than 50 p. cent at 24 hours and 48 hours, which correlated with decreased expression of 2 STAT3 target genes (Bcl-XL and c-Myc). These results suggest that STAT3 protein has a major role in the oncogenic process of nasal-type NK cell lymphomas, and may thus represent a promising therapeutical target.
- Published
- 2007
42. High Hyperdiploidy and t(12; 21) (p13; q22) Translocation Is Not a so Rare Association: A Report of 4 Cases in the Experience of Armand Trousseau Hospital (Paris)
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Judith Landman-Parker, Christine Perot, Marie-France Portnoï, Guy Leverger, Francoise Bellmann, Paola Ballerini, Anne Auvrignon, Marie-Dominique Tabone, Dalila Adjaoud, Luc Douay, Jacqueline Van Den Akker, and Stéphanie Haouy
- Subjects
Pathology ,medicine.medical_specialty ,Immunology ,Chromosome ,Aneuploidy ,Chromosomal translocation ,Karyotype ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Molecular biology ,Chromosome abnormality ,medicine ,Hyperdiploidy ,Trisomy ,Chromosome 21 - Abstract
INTRODUCTION. Since the discovery of the cryptic t(12; 21) translocation, many secondary genetic abnormalities have been described in association with TEL-AML1 fusion gene. Extensive studies of karyotypes in a few series of TEL-AML1 positive leukaemia revealed a heterogeneous pattern of chromosomal abnormalities. Numerical and structural abnormalities are often present together. The modal chromosome number does not exceed 49 so that high hyperdiploidy and TEL-AML1 fusion are so far considered mutually exclusives. Here we reported that such association is found in a small group of patients and that relapse may still occur in those patients despite the good prognostic impact commonly attributed to each lesion. PATIENTS and METHODS. Between April 1994 and April 2006, 105 children were consecutively diagnosed with TEL-AML1 positive B-ALL. TEL-AML1 expression was detected by RT-PCR in Bone Marrow (BM) diagnostic samples and the t(12;21) explored by FISH with LSI TEL-AML1 dual-color probe (Vysis) in cases with low level of fusion gene expression or lacking molecular study. Conventional cytogenetics with G and R banding was performed on BM cells after overnight and 24 hours culture RESULTS and DISCUSSION. We detected numerical and/or structural abnormalities in 82/105 (78%) of the karyotypes. Aneuploidy alone was found in 4/105(3.8%): two cases had an extra chromosome 21, one an extra chromosome 10 and one lost 1 chromosome X. Structural abnormalities alone were present in 18/105 (17.1%) and up to 58 /105 (55.2%) presented both. The most frequent structural change was observed on chromosome 12p13 (50% of all structural abnormalities) whereas the most frequent chromosome gain was +21 (14%) and +10 (6.6%). Interestingly, in four cases (3.8%) we detected high hyperdiploidy with classical supplementary chromosomes; in two of these cases a partial trisomy of chromosome 1 was also present. Karyotypes are presented: Pt1: 52,XX,+10,+16,+18,+21,+21,+22 [22]/46, XX [4] Pt 2: 54,XY,+X,+4,+ 6?[del(6q)],+9,+14,+15,+18,+21,+21,-22 [17]/46,XY [3] Pt 3: 55,XX,+X,dup(1)(q21q31),+4,+6,+10,+14,+17,+18,+21,+21 [1]/56,idem+19 [4]/56,idem,+14 [5] / 46,XX [10] Pt 4: 7,XX,+der(X)(t(X;1)(q26;q12),+4,+5,+6,+8,+10,+14,+17,+18,+21,+21 [17]/46,XX [3] Pt1 present CNS relapse at 24 months follow up without involvement of BM which tested negative for TEL-AML1 expression. Patients 2,3,4 are in continuous complete remission at respectively 6, 6 and 4 years follow up. Since t(12;21) translocation and high hyperdiploidy seem to be primary events in leukaemia our observation raises the question if the two lesions coexist in a same cell or are independent events in different clones. In two cases (Pt 3,4), FISH analysis indicated the presence of TEL-AML1 gene fusion in 7% and in 5% of nuclei respectively. The distribution and the number of the signals on AML1 locus let think that t(12;21) occurred independently from hyperdiploidy. Microarrays studies have revealed distinct gene expression signatures associated to TEL-AML1 fusion and hyperdiploidy over 50 chromosomes, our observation points out the existence of cases which could be difficult to assign to one or the other of these classes.
- Published
- 2006
43. Incidence of Ex Vivo Expansion on the Capacity of Cord Blood Graft To Generate Immune Cells: Rational for Co-Infusion of Expanded and Non Expanded Fractions?
- Author
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Ladan Kobari, Luc Douay, Michelle Rosenzwajg, Marie-Catherine Giarratana, and Jean Claude Gluckman
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medicine.medical_treatment ,Immunology ,CD34 ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Cell biology ,Immune system ,Cytokine ,Cord blood ,medicine ,Lymphopoiesis ,Myelopoiesis ,Progenitor cell ,CD8 - Abstract
The aim of our study was to determine whether ex vivo expansion of umbilical cord blood (UCB) progenitor cells induces changes in their capacity to generate immune cells. CD34+ CB cells were cultured for 14 days with SCF, FLT3-l, TPO and G-CSF, inducing a total cells, CD34+ and LTC-ICs increase of 1500, 120 and 8 fold respectively. Non expanded (d0) and 14-day expanded (d14) CD34+ cells were compared for their capacity to produce T lymphocytes (TLs) using the fetal thymus organ culture system and DCs generated from d0 and d14 CD34+ cells were compared for their differentiation, phenotype and function. Total percentages of CD4+, CD4+CD8− and CD4+CD8+ TLs obtained from d0 and d14 CD34+ cells were comparable. In both fractions, most of the CD4+ T cells co-expressed iCD3 but a lower proportion of d14 derived TLs expressed sCD3. However, there was no significant difference between d0 and d14 derived TLs in term of Vb chain representation, all TCR-Vb chains examined being represented in each case. These data indicate that d0 and d14 CD34+ cells have a similar capacity to generate TLs and that expansion does not induced any skewing of the TCR-Vb repertoire. D0 and d14 CD34+ cells were next cultured with SCF, FL, GM-CSF and TNF-a to compare their capacity to differentiate into DCs. Similar percentages of CD1a+ DCs expressing the same levels of HLA-DR and co stimulatory molecules were obtained. DCs derived from d14 CD34+ cells were less potent to stimulate allogeneic TLs, but the pattern of cytokines produced by stimulated TLs was similar and no shift towards a predominant Th1 or Th2 response was observed. Moreover, in spite of a quantitative increase (15 fold) related to the CD34 pool amplification, we observed a decreased capacity (13-fold) of d14 cells to generate DCs compared to d0 CD34+ cells. Overall, these results indicate that ex vivo expansion of CD34+ cells doesn’t induce any major modification in T Lymphopoiesis capacity while alters somehow the capacity of the graft to generate DCs. We discuss in the context of UCB transplantation, the putative interest of co-infusion of expanded and non expanded fractions in view of improving myelopoiesis in the graft without subverting the immune reconstitution.
- Published
- 2004
44. Massive and Selective Ex Vivo Generation of Matured and Functional Human Red Blood Cells (RBC) from Hematopoietic Stem Cells of Diverse Origins: Towards the New Concept of 'Cultured RBC'
- Author
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Michael C. Marden, David Chalmers, Marie-Catherine Giarratana, Henri Wajcman, Ladan Kobari, Hélène Lapillonne, Laurent Kiger, Luc Douay, and Thérèse Cynober
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education.field_of_study ,Stromal cell ,Immunology ,Population ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Molecular biology ,Haematopoiesis ,medicine.anatomical_structure ,Cord blood ,medicine ,Erythropoiesis ,Bone marrow ,Stem cell ,education ,Ex vivo - Abstract
We report a technological approach permitting, for the first time, the massive (up to 2x106-fold cell expansion) and selective (100%) ex vivo production of mature RBCs (cRBCs) starting from CD 34+ cells from peripheral blood (PB), bone marrow (BM) or cord blood (CB) into mature red cells in three steps: firstly, cell proliferation and erythroid differentiation were induced in serum free media supplemented with SCF, IL-3 and Epo for 8 days. Secondly, cells were co-cultured with additional Epo alone on either the murine MS-5 stromal cell line or human mesenchymal cells for 3 days. In the third step, all exogenous factors were withdrawn and cells were incubated on a simple stroma for 4 to 10 days. These cultured erythroid cells (reticulocytes and mature RBCs) displayed characteristics identical to those of native cells, in terms of MCV, MCH, MCHC, enzyme content (G6PD and PK) and deformability. The nature of the Hb produced depended on both the origin of the CD34+ cells and the culture conditions. cRBCs derived from PB or adult BM contained adult Hb (95±1%) whereas cRBCs derived from CB contained essentially HbF (64±13%). As for native RBCs, Hb was able to fix and release oxygen. CFSE-labelled-reticulocytes ex vivo generated from leukapheresis were injected into NOD-SCID mice. The transfused reticulocytes were found in the circulation to the same extent as native RBCs and fully matured into RBCs. This methodology is applicable for fundamental analysis of the mechanisms of terminal erythropoiesis and hemoglobin synthesis. Moreover, large scale cRBCs production could be possible with such a protocol. It can therefore be extrapolated to a wide range of clinical applications in the field of gene therapy, infectious diseases and particularly transfusion medicine with a pointed interest for the generation of a cell population homogeneous in age, thus achieving the new concept of cultured RBCs transfusion.
- Published
- 2004
45. Failure of bone marrow cryopreservation in chronic granulocytic leukemia: relation to excessive granulo-macrophagic progenitor pool
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G. Duhamel, Stachowiak J, M. Lopez, Salmon C, Laporte Jp, Norbert-Claude Gorin, Marie-Catherine Giarratana, Luc Douay, and A. Nauman
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Cell Survival ,Bone Marrow Cells ,Biology ,Transplantation, Autologous ,Cryopreservation ,Andrology ,Colony-Forming Units Assay ,Freezing ,medicine ,Humans ,Progenitor cell ,Progenitor ,Bone Marrow Transplantation ,Macrophages ,Graft Survival ,Hematopoietic Stem Cell Transplantation ,Cell Biology ,Aplasia ,Chronic granulocytic leukemia ,medicine.disease ,Hematopoietic Stem Cells ,Hematopoiesis ,Haematopoiesis ,medicine.anatomical_structure ,Leukemia, Myeloid ,Immunology ,Bone marrow ,Stem cell ,Granulocytes - Abstract
Autologous bone marrow transplantation (ABMT) in chronic granulocytic leukemia (CGL) aims at reversing the acute or acceleration phases by injection of stem cells collected during the chronic phase. This study was designed to explain an unusual rate of delayed engraftment (50%) in our experience of ABMT in CGL patients. We investigated all the factors possibly responsible for abnormal perpetuation of aplasia following infusion of cryopreserved marrow stem cells. The study of CFU-gm recovery in 41 bags of frozen marrow from 25 patients revealed an overall deficiency with a mean CFU-gm recovery of 55 +/- 38% in CGL patients versus 73 +/- 15% in the control group (p less than 0.001). Our data also showed an inverse linear relation (r = -0.40, p less than 0.05) between CFU-gm concentration and recovery after freezing. A good CFU-gm recovery (greater than or equal to = 50%) was observed in 70% of cases when the concentration was less than 3700 CFU-gm/ml as compared to 30% of cases when the concentration was over 3700 CFU-gm/ml (p less than 0.001). The lack of improvement by diluting rich CFU-gm marrows to reduce CFU-gm concentration/ml, as well as the absence of relationship between CFU-gm recovery after freezing and nucleated cells concentration, suggest a particular fragility of CGL stem cells to freezing, probably related to their excessive amplification. At the present time, we strongly recommend that the highest possible dose of progenitor cells be cryopreserved, preferably at a low concentration, in patients with CGL, and particular attention devoted to the freezing procedure in each individual patient, with numerous appropriate efficiency tests.
- Published
- 1986
46. Molecular epidemiological study in pediatric acute myeloid leukemia
- Author
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Luc Douay, Christine Perot, Jean-Luc Laï, Hughes Leroy, Françoise Mazingue, Judith Landman-Parker, Guy Leverger, Claude Preudhomme, Hélène Lapillonne, S. Lejeune-Dumoulin, A.S. Goetgheluck-Gadenne, Paola Ballerini, Mircea Adam, and Anne Auvrignon
- Subjects
medicine.medical_specialty ,Univariate analysis ,Pathology ,Mutation ,Monosomy ,Immunology ,Cytogenetics ,Cell Biology ,Hematology ,Biology ,medicine.disease_cause ,medicine.disease ,Biochemistry ,Gastroenterology ,law.invention ,law ,Internal medicine ,Epidemiology ,CEBPA ,medicine ,Polymerase chain reaction ,Rare disease - Abstract
It is hypothesized that AML arises from two cooperative types of mutations: type I mutations mainly induce proliferation and type II mutations involved in the maturation arrest. AML is a rare disease in children and few molecular data are available on pediatric AML. We therefore studied N-RAS, K-RAS, FLT3-ITD, FLT3 , C-KIT mutations (type I), and CEBPA mutations (type II) as well as FLT3, EVI-1 and WT1 gene expression in 77 de novo AML. Patients and methods: All the patients (aged 1 month-17 years, median age: 6.9 years, male/female ratio 1.26) were treated for de novo AML between 1995 and 2003 in two French institutions and prospectively enrolled in LAM91, LAM01 and APLs French collaborative protocols. According to the FAB classification the repartition was: M0:6.5%, M1: 5.2%, M2: 22%, M3: 13%, M4 :14.3%, M5 :30%, M7: 6.5% and unclassified :2.5%. Cytogenetics features according to the MRC classification were favorable, intermediate or poor in 25% (t(8;21) n=5; t(15,17) n=8, inv(16) n=5), 65% (normal n=20 and 11q23 abnormalities n=15) and 10% (−7, n=4) respectively. With a median follow-up of 26 months (range 2–98 months), Complete Remission was obtained in 92% (71/77) of patients, OS was 71% and EFS 61%. CEBPA, N-RAS, K-RAS, C-KIT and FLT3 mutations detection was performed by direct sequencing. FLT3, EVI-1 and WT1 transcripts were quantified by RQ-PCR. Results: (1) Frequency of N-RAS and K-RAS mutations were 11% (8/75) and 16% (12/75) respectively. RAS-mutated patients belonged to favorable (30%), intermediate (60%) and poor (10%) cytogenetic subgroups. In univariate analysis only N-RAS mutations is associated with adverse outcome (OS 37% vs 79%, p Conclusion: In total, 48% of de novo AML in children had a mutation in N-RAS, K-RAS, FLT3-ITD, FLT3 Asp835, C-KIT or CEBPA with a high frequency of RAS mutations (27%) compared to adult AML and a significantly bad survival. Additional gene expression quantification of EVI-1, FLT3 and WT1 allows MDR detection in 95% of patients.
47. WT1 overexpression after induction therapy in children AML is associated with higher risk of relapse
- Author
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Christine Perot, Sylvie Fasola, Cyril Flamant, Paola Ballerini, Anne Auvrignon, Annick Blaise, Francoise Mazingue, Claude Preudhomme, Guy Leverger, Hugues Leroy, Luc Douay, Hélène Lapillonne, and Judith Landman-Parker
- Subjects
medicine.medical_specialty ,Subsequent Relapse ,Tumor suppressor gene ,business.industry ,Immunology ,Cytogenetics ,Cell Biology ,Hematology ,Biochemistry ,Minimal residual disease ,Gastroenterology ,law.invention ,law ,Median follow-up ,Internal medicine ,Induction therapy ,medicine ,Relapse risk ,business ,Polymerase chain reaction - Abstract
The Wilms’ s tumor gene (WT1) is a tumor suppressor gene highly expressed in most acute leukemias. To determine whether WT1 gene expression is a valuable and informative marker for minimal residual disease in pediatric AML, we quantified WT1 transcript amount by RQ-PCR in 92 de novo AML and 20 normal controls. The WT1 transcripts obtained were normalized with respect to the number of TBP transcripts and expressed as WT1 copy numbers by the ratio WT1/TBPx1000. The WT1 levels were extremely low in normal controls, and the median number of WT1 copies was 10 (range 4–30 ). A level above 50 copies was considered as significant. All the patients (aged 2 months-18 years, median age:5.9 years, male/female ratio 0.87) were treated for de novo AML between 1995 and 2003 in two French institutions and enrolled in LAM91, LAM01 and APLs French collaborative protocols. According to the FAB classification the repartition was: MO 5.4%, M1 4.3%, M2 18.5%, M3 14.1%, M4 and M4Eo 12%, M5 33.6 %, M7 10.9% and unclassified 1%. Cytogenetics features according to the MRC classification were favourable, intermediate or poor in 27% (23/83), 59% (49/83) and 13% (11/83) respectively. With a median follow up of 24 months (range 8–97months) OS was 75± 8% and EFS 60 ± 6%. At diagnosis WT1 overexpression was detected in 78.3% (72/92) with a median copy number of 2231 (range 50-429200). The WT1 values were significantly higher (p=0.02) in M2-FAB subtype and lower (p=0.01) in M5-FAB subtype while no correlation was found with WBC count or cytogenetic abnormalities. WT1 quantification for MRD was evaluable in 41/72 pts and positive in 9/32 at D40-50, 5/25 at M3-M5, 5/7at M6-M8. At least one analysis above 50 copies after induction therapy is associated with a significant risk of subsequent relapse 8/11 vs 8/30 ( p=0.007) RR=22 (IC 95%:46–118) and death 10/14 vs 3/30 (p=0.001) RR=7.3 (IC95%: 1–34). Although retrospective analysis may include bias, we conclude that WT1 is a useful and informative molecular marker for MRD in pediatric AML and performed as prospective analysis in ELAM02 protocol.
48. Autologous bone marrow transplantation using marrow incubated with Asta Z 7557 in adult acute leukemia
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Jean-Yves Mary, Jean-Philippe Laporte, Stachowiak J, Philippe Aegerter, Albert Najman, Norbert-Claude Gorin, Salmon C, R. David, Marc Lopez, and Luc Douay
- Subjects
Adult ,Male ,medicine.medical_specialty ,Cyclophosphamide ,Adolescent ,Erythroblasts ,Immunology ,Cell Separation ,Gastroenterology ,Biochemistry ,Colony-Forming Units Assay ,chemistry.chemical_compound ,Mafosfamide ,Internal medicine ,medicine ,Humans ,Bone Marrow Transplantation ,Acute leukemia ,Clinical Trials as Topic ,Leukemia ,business.industry ,Stem Cells ,Cell Biology ,Hematology ,Total body irradiation ,Middle Aged ,medicine.disease ,Surgery ,Haematopoiesis ,chemistry ,Liver ,Toxicity ,Female ,Stem cell ,business ,medicine.drug ,Granulocytes - Abstract
The sensitivity of human myeloblastic leukemic (CFU-L) and normal hemopoietic stem cells (CFU-GM and BFU-e) to Asta Z 7557 (INN Mafosfamide) was studied with regard to autologous bone marrow transplantation (ABMT) with cleansed marrow for consolidation therapy in adult patients with acute leukemia (AL) in remission. Establishment of the dose-response curves for CFU-GM (n = 37), BFUe (n = 11), and myeloblastic CFU-L (n = 9) demonstrated a wide range of sensitivity from patient to patient for all three progenitors. Whereas CFU-L, CFU- GM, and BFU-e grown in semisolid cultures disclosed similar sensitivities to Asta Z 7557, long-term culture (LTC) studies (n = 41) indicated a higher resistance of early progenitors. In an effort to achieve a maximum tumor cell kill and yet spare a sufficient amount of normal stem cells to ensure consistent engraftment, we defined the optimal dose for marrow cleansing as the dose sparing 5% CFU-GM (LD95). This dose was established from a preincubation test (PIT) realized on a 10-mL marrow aspirate taken 15 days before marrow collection in each individual patient. Twenty-four adult patients while in remission of AL (20 in complete remission, four in partial remission) were consolidated by cyclophosphamide 60 mg/kg X 2 and total body irradiation at 10 Gy followed by ABMT with marrow cleansed by Asta Z 7557 according to the specification described above. Patients were divided in two groups: group 1, unfavorable prognosis (11 patients); group 2, standard prognosis [13 patients in first complete remission (CR)]. All patients engrafted on leukocytes (median day for recovery to 10(9)/L: day 30), patients with ALL recovered faster than patients with ANL (median day 19 v 34). Similarly, recovery of platelets to 50.10(9)/L occurred sooner in patients with ALL (median day 67, range day 23 through 90) whereas three patients with acute nonlymphoblastic leukemia (ANLL) in group 2 had to be supported with platelet transfusions for more than one year. In group 1, six patients had recurrent tumor within six months; three patients died from toxicity with no evidence of tumor. Two patients are still disease-free with a short follow-up (nine and ten months). In group 2, two patients died from toxicity with no evidence of leukemia three and 16 months post-ABMT. One patient with a M5 ANLL and one patient with ALL relapsed at six and 15 months, respectively. Nine patients have remained in CR or are disease-free with a median follow-up of 22 months.(ABSTRACT TRUNCATED AT 400 WORDS)
49. A prospective study of the value of bone marrow erythroid progenitor cultures in polycythemia
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Duhamel G, Jean Philippe Laporte, Albert Najman, François M. Lemoine, Claude Baillou, Stachowiak J, Philippe Aegerter, Norbert Claude Gorin, Georges Boffa, and Luc Douay
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Adult ,Male ,Pathology ,medicine.medical_specialty ,Secondary Polycythemia ,Erythroid progenitor ,Immunology ,Polycythemia ,Biochemistry ,Group A ,Gastroenterology ,Group B ,Polycythemia vera ,Bone Marrow ,hemic and lymphatic diseases ,Internal medicine ,Humans ,Medicine ,Erythropoiesis ,Prospective Studies ,Prospective cohort study ,Erythropoietin ,Cells, Cultured ,Aged ,Heterogeneous group ,business.industry ,Cell Biology ,Hematology ,Middle Aged ,medicine.disease ,stomatognathic diseases ,medicine.anatomical_structure ,Female ,Bone marrow ,business - Abstract
The diagnostic value in polycythemia of the presence of endogenous erythroid colonies derived from bone marrow cells (EECs) was assessed in a prospective study on 108 patients referred for polycythemia (Hb greater than g/dL in men, greater than 16 g/dL in women) with normal plasma volume by comparison with the standard criteria, the bone marrow grade, and the serum erythropoietin (Epo) level. Total red cell volume (TRCV) was high (greater than 36 mL/kg in men, 32 mL/kg in women) in 87 cases (group A) and slightly increased in 21 cases (group B). Standard criteria were applicable in 63 of 108 cases (57%); 46 were PV and 17 were secondary polycythemia (SP). Standard criteria were nonapplicable in 45 cases. EECs were present in 65 cases (60%) with a ratio of EEC/Epo-stimulated colonies of 39.5% +/- 18% (extremes 10% to 80%). EECs were noted in 43 of 46 polycythemia vera (PV) and 0 of 17 SP. Among the 45 unclassified cases, EECs were noted in 22: 18 of 29 cases from group A (10 with 2 major and 1 minor criteria; 8 with 2 major criteria) and 4 of 16 cases from group B (with variable standard criteria, 2 belonging to a PV family). In group A, there was a positive significant correlation between EECs and the presence of two major and 1 minor criteria (P less than .0001). In group B, there was a positive significant correlation between EECs and the presence of at least 1 major criterion and 2 minor criteria or a family background (P less than .0001). The unclassified polycythemias with EECs in the bone marrow are characterized by a bone marrow grade and a mean serum Epo level not different from that of patients with PV and an active course of the disease. The unclassified polycythemias without EECs in the bone marrow are a heterogeneous group corresponding in some cases to SPs of unknown origin (slightly increased bone marrow grade and/or high serum Epo level), and in others cases to spurious polycythemias (normal bone marrow grade and/or normal Epo level). In conclusion, EECs were of great value in differentiating PV from SP (P less than .001), and in allowing the diagnosis of PV in the absence of all the standard criteria even when TRCV was slightly increased. In our study, EEC improved the classification of polycythemia by 22%. The recommended diagnostic steps for the evaluation of polycythemia must be reconsidered.
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