Figure 4.
The role of IL-3 in EFS-ADA lentiviral transduction of human cord blood CD34+ cells and engraftment in NSG mice. Human cord blood CD34+ cells were transduced with LV EFS-ADA (3 × 107 TU/ml) in medium with recombinant human cytokines SCF/ckit ligand, flt-3 ligand, and thrombopoietin (TPO), with or without interleukin-3 (IL-3). (a-d) In vitro: (a) transduced cells cultured for 14 days in vitro under myeloid differentiation conditions and analyzed for VCN (14d VCN) (N.S, not significant). (b,c) Transduced CD34+ cells were grown in colony-forming unit (CFU) assay in methylcellulose and assayed after 2 weeks. Colony (b) enumeration and (c) types formed by CD34+ cells in CFU assay. (d,e) CFU were harvested and DNA analyzed by qPCR for VCN (d) Transduction efficiency was measured by the percentage of colonies positive for vector sequence by PCR for the human ADA cDNA (%PCR (+) CFU). (e) VCN was quantified in DNA extracted from individual CFU by qPCR (*P value = 0.001). (f-h) In Vivo: other portions of the transduced CD34+ cells were transplanted into NSG mice and analyzed after 4 months for engraftment of human cells based on FACS analysis of huCD45 expression and for VCN by qPCR. Engraftment of human cells in (f) bone marrow, thymus (when present) and spleen by FACS of tissue cell suspensions immunostained with anti-human CD45 (%hCD45+). (g) EFS-ADA VCN in bone marrow, thymus (when enough cells were available for analysis; total n = 3),and spleen. (h) Immunophenotypic analysis of human CD45+ cells in NSG bone marrow (CD34+ and CD33+), thymus (CD4−/CD8− double-negative (DN), CD4+/CD8+ double-positive (DP), CD4+ single-positive (SP-4) and CD8+ single-positive (SP-8)) and spleen (CD19+ and CD3+).