title: Deficiency of ribosomal protein S19 in CD34+ cells generated by siRNA blocks erythroid development and mimics defects seen in Diamond-Blackfan anemia.
abstract: Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein S19 (RPS19) gene. To study effects of RPS19 deficiency in hematopoiesis we transduced CD34(+) umbilical cord blood (CB) and bone marrow (BM) cells with 3 lentiviral vectors expressing small interfering RNA (siRNA) against RPS19 and 1 scrambled control vector. All vectors also express green fluorescent protein (GFP). Transduction with the siRNA vectors reduced RPS19 mRNA levels to various degrees, which resulted in erythroid defects, correlating to the degree of RPS19 down-regulation, and was rescued by expression of an siRNA-resistant RPS19 transcript. Erythroid colony formation capacity conjointly decreased with RPS19 levels in CD34(+) CB and BM cells. In liquid culture supporting erythroid differentiation, RPS19-silenced as well as DBA patient CD34(+) cells exhibited reduced proliferative capacity and impaired erythroid differentiation resulting in fewer erythroid colony-forming units (CFU-Es). When assaying myeloid development, a less pronounced influence on proliferation was seen. This study shows for the first time that RPS19 silencing decreases the proliferative capacity of hematopoietic progenitors and leads to a defect in erythroid development.
authors.affiliation: Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine and The Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Flygare
authors.forename: Thomas
authors.initials: T
authors.lastname: Kiefer
authors.forename: Koichi
authors.initials: K
authors.lastname: Miyake
authors.forename: Taiju
authors.initials: T
authors.lastname: Utsugisawa
authors.forename: Isao
authors.initials: I
authors.lastname: Hamaguchi
authors.forename: Lydie
authors.initials: L
authors.lastname: Da Costa
authors.forename: Johan
authors.initials: J
authors.lastname: Richter
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Davey
authors.forename: Hans
authors.initials: H
authors.lastname: Matsson
authors.forename: Niklas
authors.initials: N
authors.lastname: Dahl
authors.forename: Maciej
authors.initials: M
authors.lastname: Wiznerowicz
authors.forename: Didier
authors.initials: D
authors.lastname: Trono
authors.forename: Stefan
authors.initials: S
authors.lastname: Karlsson
mesh_terms: D029503:Anemia, Diamond-Blackfan / Q000097:blood* / Q000473:pathology; D015703:Antigens, CD / Q000096:biosynthesis; D018952:Antigens, CD34 / Q000096:biosynthesis*; D000944:Antigens, Differentiation, B-Lymphocyte / Q000096:biosynthesis; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000166:cytology; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D002469:Cell Separation; D003001:Cloning, Molecular; D015536:Down-Regulation; D015672:Erythroid Precursor Cells / Q000378:metabolism; D005434:Flow Cytometry; D020868:Gene Silencing; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006410:Hematopoiesis; D006801:Humans; D007091:Image Processing, Computer-Assisted; D016086:Lentivirus / Q000235:genetics; D008969:Molecular Sequence Data; D010641:Phenotype; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D011990:Receptors, Transferrin; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000172:deficiency* / Q000502:physiology*; D012270:Ribosomes / Q000378:metabolism; D013997:Time Factors; D019076:Transgenes; D014470:Umbilical Cord / Q000166:cytology
chemical_list: D015703:Antigens, CD; D018952:Antigens, CD34; D000944:Antigens, Differentiation, B-Lymphocyte; C068126:CD71 antigen; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011990:Receptors, Transferrin; D012269:Ribosomal Proteins; C040343:ribosomal protein S19; D049452:Green Fluorescent Proteins