title: Optimal conditions for lentiviral transduction of engrafting human CD34+ cells.
abstract: Cytokines are required for γ-retroviral transduction of human CD34+ cells. However, cytokines may reduce engraftment of CD34+ cells and may not be necessary for their lentiviral transduction. We sought to optimize transduction and engraftment of human CD34+ cells using lentiviral vectors. Single 24 h transduction of human CD34+ cells with human immunodeficiency virus type 1 (HIV1)-based lentiviral vectors in media containing stem cell factor (SCF), FMS-like tyrosine kinase 3 (FLT3) ligand, thrombopoietin (each 100 ng ml⁻¹) and 10% fetal bovine serum was compared with various cytokine conditions during ex vivo culture and assayed using humanized xenograft mice for 6 months after transplantation. Serum-free media improved transduction efficiency of human CD34+ cells. Interleukin-3 (20 ng ml⁻¹) had little effect on transduction efficiency or engraftment. Threefold higher cytokine mixture (each 300 ng ml⁻¹) reduced engraftment of CD34+ cells. SCF alone (100 ng ml⁻¹) proved insufficient for maintaining engraftment ability and reduced transduction efficiency. Short-term prestimulation had little effect on transduction efficiency or engraftment, yet 24 h prestimulation showed higher transduction efficiency, higher gene expression levels and lower engraftment. In summary, 24 h prestimulation followed by single 24-h lentiviral transduction in serum-free media with SCF, FLT3 ligand and thrombopoietin yields high transduction efficiency to engrafting human CD34+ cells, and is applicable in human clinical gene therapy trials.
authors.affiliation: Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institute-NHLBI/National Institute of Diabetes and Digestive and Kidney Diseases-NIDDK, National Institutes of Health-NIH, Bethesda, MD 20892, USA.
authors.forename: N
authors.initials: N
authors.lastname: Uchida
authors.forename: M M
authors.initials: MM
authors.lastname: Hsieh
authors.forename: J
authors.initials: J
authors.lastname: Hayakawa
authors.forename: C
authors.initials: C
authors.lastname: Madison
authors.forename: K N
authors.initials: KN
authors.lastname: Washington
authors.forename: J F
authors.initials: JF
authors.lastname: Tisdale
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism*; D016895:Culture Media, Serum-Free; D005822:Genetic Vectors; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology*; D016086:Lentivirus / Q000235:genetics*; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D023421:Models, Animal; D019089:Stem Cell Factor / Q000276:immunology; D013926:Thrombopoietin / Q000276:immunology; D014161:Transduction, Genetic; D014183:Transplantation, Heterologous; D051941:fms-Like Tyrosine Kinase 3 / Q000276:immunology
chemical_list: D018952:Antigens, CD34; D016895:Culture Media, Serum-Free; D007377:Interleukin-3; D019089:Stem Cell Factor; D013926:Thrombopoietin; D051941:fms-Like Tyrosine Kinase 3