Deficiency of ribosomal protein S19 in CD34+ cells generated by siRNA blocks erythroid development and mimics defects seen in Diamond-Blackfan anemia

• 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.

Ämnesord

MEDICIN OCH HÄLSOVETENSKAP  -- Klinisk medicin -- Hematologi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Clinical Medicine -- Hematology (hsv//eng)

Nyckelord

Anemia; Diamond-Blackfan/*blood/pathology

Antigens; CD/biosynthesis

Antigens; CD34/*biosynthesis

Antigens; Differentiation; B-Lymphocyte/biosynthesis

Base Sequence

Blotting; Northern

Blotting; Western

Bone Marrow Cells/cytology

Cell Differentiation

Cell Line

Cell Proliferation

Cell Separation

Cloning; Molecular

Down-Regulation

Erythroid Progenitor Cells/metabolism

Flow Cytometry

Gene Silencing

Genetic Vectors

Green Fluorescent Proteins/chemistry/metabolism

Hematopoiesis

Humans

Image Processing; Computer-Assisted

Lentivirus/genetics

Molecular Sequence Data

Phenotype

RNA; Messenger/metabolism

RNA; Small Interfering/*metabolism

Receptors; Transferrin

Reverse Transcriptase Polymerase Chain Reaction

Ribosomal Proteins/*deficiency/*physiology

Ribosomes/metabolism

Time Factors

Transgenes

Umbilical Cord/cytology

MEDICINE

MEDICIN

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