title: Efficient retroviral transduction of human B-lymphoid and myeloid progenitors: marked inhibition of their growth by the Pax5 transgene.
abstract: We applied a coculture system for the genetic manipulation of human B-lymphoid and myeloid progenitor cells using murine bone marrow stromal cell support, and investigated the effects of forced Pax5 expression in both cell types. Cytokine-stimulated cord blood CD34+ cells could be transduced at 85% efficiency and 95% cell viability by a single 24-h infection with RD114-pseudotyped retroviral vectors, produced by the packaging cell line Plat-F and bicistronic vector plasmids pMXs-Ig, pMYs-Ig, or pMCs-Ig, encoding EGFP. Infected CD34+ cells were seeded onto HESS-5 cells in the presence of stem cell factor and granulocyte colony-stimulating factor, allowing the extensive production of B progenitors and granulocytic cells. We examined the cell number and CD34, CD33, CD19, and CD20 lambda and kappa expressions by flow cytometry. Ectopic expression of Pax5 in CD34+ cells resulted in small myeloid progenitors coexpressing CD33 and CD19 and inhibited myeloid differentiation. After 6 weeks, the number of Pax5-transduced CD19+ cells was 40-fold lower than that of control cells. However, the expression of CD20 and the kappa/lambda chain on Pax5-transduced CD19+ cells suggests that the Pax5 transgene may not interfere with their differentiation. This report is the first to describe the effects of forced Pax5 expression in human hematopoietic progenitors.
authors.affiliation: Division of Molecular Therapy, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.
authors.forename: Rieko
authors.initials: R
authors.lastname: Sekine
authors.affiliation: Division of Cellular Therapy, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Kitamura
authors.affiliation: Department of Industrial Science and Technology, Science University of Tokyo, Noda, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Tsuji
authors.affiliation: Division of Molecular Therapy, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan. a-tojo@ims.u-tokyo.ac.jp.
authors.forename: Arinobu
authors.initials: A
authors.lastname: Tojo
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D051379:Mice; D023461:Myeloid Progenitor Cells / Q000166:cytology* / Q000378:metabolism*; D051757:PAX5 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D054448:Precursor Cells, B-Lymphoid / Q000166:cytology* / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics*; D019076:Transgenes / Q000235:genetics*
chemical_list: D018952:Antigens, CD34; D051757:PAX5 Transcription Factor; C497281:PAX5 protein, human