Figure 7
Figure 7. HSPC in vivo transduction in a humanized mouse model. (A) CD46 MFI on human CD34+ and CD34+/CD38− cells derived from umbilical cord blood MNCs. (B-D) In vivo studies in humanized mice. NOG mice received whole body irradiation and were transplanted with human CD34+ cells. Six weeks after transplantation, successful engraftment was confirmed by huCD45 flow cytometry of PBMCs. Animals were then mobilized and injected with HD-Ad–SB + HD-Ad–GFP. (B) Mobilization of human HSPCs. PBMCs were collected 90 minutes after AMD3100. PBMCs were plated in CFU assays in the presence of human or murine cytokines. Total CFU were enumerated 12 days after plating. N = 2. Differences between mobilized and nonmobilized animals were not statistically significant after unpaired Student t tests. (C) GFP expression in total human CD45+ cells and in HSPCs (CD34+ or c-Kit+ cells) in the BM, spleen, and PBMCs 3 days after HD-Ad–SB + HD-Ad–GFP injection into mobilized mice. N = 2. **P < .01 after one-way ANOVA with Bonferroni posttesting. (D) GFP expression in human (hCD45+) cells in the BM and spleen at day 3 (N = 4) and week 4 (N = 7) after in vivo transduction with HD-Ad–SB + HD-Ad–GFP. Values represent mean ± SD. ***P < .001 after one-way ANOVA with Bonferroni posttesting. (E-F) Lineage composition of hematopoietic tissues and transgene expression in hematopoietic lineages following HSPC in vivo transduction. Humanized NOG mice were mobilized and injected with HD-Ad–SB + HD-Ad–GFP as before. Animals were euthanized at 3 days (n = 2) or 4 weeks (n = 3) after transduction, and expression of GFP and lineage surface markers was assessed via flow cytometry. (E) Expression of hematopoietic lineage surface markers after HSPC in vivo transduction in the BM, spleen, and peripheral blood. An unmobilized, untreated, humanized NOG animal was used as control (mock). Shown are mean ± SD. (F) GFP expression in hematopoietic lineages at 3 days and 4 weeks after HSPC in vivo transduction. Shown are mean ± SD. *P < .05 following two-way ANOVA with Bonferroni posttesting. **P < .01; ***P < .001. iso, isolated; n.s., not significant; mobil, mobilized.

HSPC in vivo transduction in a humanized mouse model. (A) CD46 MFI on human CD34+ and CD34+/CD38− cells derived from umbilical cord blood MNCs. (B-D) In vivo studies in humanized mice. NOG mice received whole body irradiation and were transplanted with human CD34+ cells. Six weeks after transplantation, successful engraftment was confirmed by huCD45 flow cytometry of PBMCs. Animals were then mobilized and injected with HD-Ad–SB + HD-Ad–GFP. (B) Mobilization of human HSPCs. PBMCs were collected 90 minutes after AMD3100. PBMCs were plated in CFU assays in the presence of human or murine cytokines. Total CFU were enumerated 12 days after plating. N = 2. Differences between mobilized and nonmobilized animals were not statistically significant after unpaired Student t tests. (C) GFP expression in total human CD45+ cells and in HSPCs (CD34+ or c-Kit+ cells) in the BM, spleen, and PBMCs 3 days after HD-Ad–SB + HD-Ad–GFP injection into mobilized mice. N = 2. **P < .01 after one-way ANOVA with Bonferroni posttesting. (D) GFP expression in human (hCD45+) cells in the BM and spleen at day 3 (N = 4) and week 4 (N = 7) after in vivo transduction with HD-Ad–SB + HD-Ad–GFP. Values represent mean ± SD. ***P < .001 after one-way ANOVA with Bonferroni posttesting. (E-F) Lineage composition of hematopoietic tissues and transgene expression in hematopoietic lineages following HSPC in vivo transduction. Humanized NOG mice were mobilized and injected with HD-Ad–SB + HD-Ad–GFP as before. Animals were euthanized at 3 days (n = 2) or 4 weeks (n = 3) after transduction, and expression of GFP and lineage surface markers was assessed via flow cytometry. (E) Expression of hematopoietic lineage surface markers after HSPC in vivo transduction in the BM, spleen, and peripheral blood. An unmobilized, untreated, humanized NOG animal was used as control (mock). Shown are mean ± SD. (F) GFP expression in hematopoietic lineages at 3 days and 4 weeks after HSPC in vivo transduction. Shown are mean ± SD. *P < .05 following two-way ANOVA with Bonferroni posttesting. **P < .01; ***P < .001. iso, isolated; n.s., not significant; mobil, mobilized.

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