title: Inhibition of Thrombopoietin/Mpl Signaling in Adult Hematopoiesis Identifies New Candidates for Hematopoietic Stem Cell Maintenance.
abstract: Thrombopoietin (Thpo) signals via its receptor Mpl and regulates megakaryopoiesis, hematopoietic stem cell (HSC) maintenance and post-transplant expansion. Mpl expression is tightly controlled and deregulation of Thpo/Mpl-signaling is linked to hematological disorders. Here, we constructed an intracellular-truncated, signaling-deficient Mpl protein which is presented on the cell surface (dnMpl). The transplantation of bone marrow cells retrovirally transduced to express dnMpl into wildtype mice induced thrombocytopenia, and a progressive loss of HSC. The aplastic BM allowed the engraftment of a second BM transplant without further conditioning. Functional analysis of the truncated Mpl in vitro and in vivo demonstrated no internalization after Thpo binding and the inhibition of Thpo/Mpl-signaling in wildtype cells due to dominant-negative (dn) effects by receptor competition with wildtype Mpl for Thpo binding. Intracellular inhibition of Mpl could be excluded as the major mechanism by the use of a constitutive-dimerized dnMpl. To further elucidate the molecular changes induced by Thpo/Mpl-inhibition on the HSC-enriched cell population in the BM, we performed gene expression analysis of Lin-Sca1+cKit+ (LSK) cells isolated from mice transplanted with dnMpl transduced BM cells. The gene expression profile supported the exhaustion of HSC due to increased cell cycle progression and identified new and known downstream effectors of Thpo/Mpl-signaling in HSC (namely TIE2, ESAM1 and EPCR detected on the HSC-enriched LSK cell population). We further compared gene expression profiles in LSK cells of dnMpl mice with human CD34+ cells of aplastic anemia patients and identified similar deregulations of important stemness genes in both cell populations. In summary, we established a novel way of Thpo/Mpl inhibition in the adult mouse and performed in depth analysis of the phenotype including gene expression profiling.
authors.affiliation: Research Group for Gene Modification in Stem Cells, LOEWE Center for Cell and Gene Therapy Frankfurt/Main and the Paul-Ehrlich-Institute, Langen, Germany; Institute of Experimental Hematology; Hannover Medical School, Hannover, Germany.
authors.forename: Saskia
authors.initials: S
authors.lastname: Kohlscheen
authors.affiliation: Institute of Experimental Hematology; Hannover Medical School, Hannover, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Wintterle
authors.affiliation: Institute of Experimental Hematology; Hannover Medical School, Hannover, Germany.
authors.forename: Adrian
authors.initials: A
authors.lastname: Schwarzer
authors.affiliation: Department of Biostatistik, Paul-Ehrlich-Institute, Langen, Germany.
authors.forename: Christel
authors.initials: C
authors.lastname: Kamp
authors.affiliation: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, ZA Leiden, The Netherlands.
authors.forename: Martijn H
authors.initials: MH
authors.lastname: Brugman
authors.affiliation: Institute of Experimental Hematology; Hannover Medical School, Hannover, Germany.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Breuer
authors.affiliation: Institute of Pathology, Hannover Medical School, Hannover, Germany.
authors.forename: Guntram
authors.initials: G
authors.lastname: Büsche
authors.affiliation: Institute of Experimental Hematology; Hannover Medical School, Hannover, Germany.
authors.forename: Christopher
authors.initials: C
authors.lastname: Baum
authors.affiliation: Research Group for Gene Modification in Stem Cells, LOEWE Center for Cell and Gene Therapy Frankfurt/Main and the Paul-Ehrlich-Institute, Langen, Germany; Institute of Experimental Hematology; Hannover Medical School, Hannover, Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Modlich
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D006410:Hematopoiesis / Q000502:physiology*; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D053628:Receptors, Thrombopoietin / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013921:Thrombocytopenia / Q000378:metabolism; D013926:Thrombopoietin / Q000378:metabolism*
chemical_list: D053628:Receptors, Thrombopoietin; D013926:Thrombopoietin