Increased EGFP marking in human hematopoietic lineages arising from rapamycin-treated and LV-transduced CD34+ cells transplanted into NSG mice. Human cord blood CD34+ cells were prestimulated for 24 hours in HSC-supportive cytokines and then transduced for 12 hours with CG-UbiC-EGFP, MOI = 25, in the presence of DMSO, 10 μg/mL rapamycin, or 20 μg/mL rapamycin. NSG mice each received 3 × 106 CD34+ cells (DMSO control or 10 μg/mL rapamycin treatment) or 2.6 × 106 CD34+ cells (20 μg/mL rapamycin treatment). Mice were sacrificed 19 weeks posttransplant. (A) Total hCD45+ engraftment levels in the bone marrow. (B) EGFP expression and MFI were measured by flow cytometry in live hCD45+ from the BM (left). LV copy number per cell as determined by qPCR (described in “Methods”) (right). (C) EGFP expression in bone marrow and splenic subsets, analyzed with the following human markers: CD34+ for HSCs, CD33+ for myeloid cells, CD19+ for B cells, and CD3+ for T cells. Cells were pregated on hCD45 for subsequent lineage analyses. Each point represents 1 mouse. For all panels, lines represent group mean and error bars represent standard deviation. *P < .05, ***P < .001, and ****P < .0001 from a parametric 2-tailed unpaired Student t test.
