Optimal conditions for lentiviral transduction of engrafting human CD34+ cells.

ABSTRACT: Cytokines are required for ?-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.

SUBMITTER: Uchida N 

PROVIDER: S-EPMC6525965 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature