Efficient retroviral transduction of human B-lymphoid and myeloid progenitors: marked inhibition of their growth by the Pax5 transgene.
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.
Sekine
Rieko
R
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.
Kitamura
Toshio
T
Division of Cellular Therapy, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
Tsuji
Takashi
T
Department of Industrial Science and Technology, Science University of Tokyo, Noda, Japan.
Tojo
Arinobu
A
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.
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*
D018952:Antigens, CD34; D051757:PAX5 Transcription Factor; C497281:PAX5 protein, human