title: Analysis of optimal conditions for retroviral-mediated transduction of primitive human hematopoietic cells.
abstract: We sought to define optimal conditions for retroviral-mediated transduction of long-lived human hematopoietic progenitors from bone marrow and peripheral blood. CD34+ cells were transduced by the LN and G2 retroviral vectors in the presence or absence of stromal support and with or without cytokine addition. After transduction, a portion of the cells was plated in methylcellulose colony-forming assay, with or without G418, to assess the extent of gene transfer into committed progenitors. The remaining cells from each experiment were transplanted into immunodeficient mice to allow analysis of transduction of long-lived progenitors. Human colony-forming cells contained within the murine bone marrow were analyzed after engraftment periods of 2 to 11 months. Cells were plated in a human-specific colony-forming assay with and without G418 to assess the extent of transduction of primitive progenitors. Individual human colonies were also analyzed by polymerase chain reaction for the presence of provirus. Bone marrow progenitors were efficiently transduced only when stroma was present, whereas mobilized peripheral blood progenitors were effectively transduced in the presence of either stroma or cytokines. Inclusion of the cytokines interleukin-3, interleukin-6, and stem cell factor did not further augment the extent of gene transfer in the presence of a stromal support layer. Additionally, human CD34+ progenitors from bone marrow or mobilized peripheral blood that had been transduced for 3 days in the absence of stroma failed to produce sustained, long-term engraftment of bnx mice. Mice transplanted with the same pools of human progenitors that had been transduced in the presence of stroma for 3 days had significant levels of human cell engraftment at the same timepoints, 7 to 11 months after transplantation. Our data show loss of long-lived human progenitors during 3-day in vitro transduction periods in the absence of stromal support. Therefore, the presence of bone marrow stroma has dual benefits in that it increases gene transfer efficiency and is essential for survival of long-lived human hematopoietic progenitors.
authors.affiliation: Childrens Hospital Los Angeles, University of Southern California School of Medicine, Department of Pediatrics 90027, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Nolta
authors.forename: E M
authors.initials: EM
authors.lastname: Smogorzewska
authors.forename: D B
authors.initials: DB
authors.lastname: Kohn
mesh_terms: D000818:Animals; D001773:Blood Cells / Q000166:cytology; D001854:Bone Marrow Cells; D002470:Cell Survival; D002478:Cells, Cultured / Q000637:transplantation / Q000821:virology; D002678:Chimera; D003114:Colony-Forming Units Assay; D003238:Connective Tissue / Q000502:physiology*; D046508:Culture Techniques / Q000379:methods*; D004351:Drug Resistance / Q000235:genetics; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics / Q000302:isolation & purification; D005839:Gentamicins / Q000494:pharmacology; D006085:Graft Survival; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology*; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000821:virology*; D006801:Humans; D007153:Immunologic Deficiency Syndromes / Q000235:genetics; D019868:Kanamycin Kinase; D051379:Mice; D008817:Mice, Mutant Strains; D008819:Mice, Nude; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012190:Retroviridae / Q000235:genetics / Q000302:isolation & purification; D014162:Transfection; D014183:Transplantation, Heterologous
chemical_list: D005839:Gentamicins; D016298:Hematopoietic Cell Growth Factors; D011994:Recombinant Proteins; C010680:antibiotic G 418; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase