Fig. 1. Generation of Hu-mice.

a Schematic of Hu-mice reconstitution. Six-week-old female NSG mice are all treated with the myelo-depleting drug (busulfan [30 mg/kg]; i.p.) 24 h before they receive purified fetal liver-derived CD34⁺ cells (1 × 10⁵; i.v.) and autologous thymus grafts (~2 mm) under the renal capsule. After day 50, mice are periodically bled (100 μl) and characterized for human immune cells by standard flow cytometry assay using fluorochrome-conjugated anti-mouse or anti-human antibodies. b Repopulation of human CD45⁺ cells in circulating blood of reconstituted mice. A representative example of mice (n = 45) after 8–12 weeks of human CD34⁺ cell injection showed increased levels of human CD45⁺ cells (brown squares; p = 0.00025) in circulating blood when compared to control non-reconstituted female NSG mice (blue squares; n = 5). c Enhanced repopulation of human lymphocytes after AAV8-hu-cytokine transgenes delivery. Significant increase in circulating human CD45⁺ cells (p = 0.022 for days 48 and 72 [closed blue circles and brown squares] and p = 0.0094 for day 112 [closed black triangles]) in mice (n = 7) that received AAV8 hu-cytokines (IL3, IL-7, and GM-CSF; 2 × 10⁹ GC/ml; i.v.; 5 days after CD34 injection; right panel) when compared to mice (n = 5) that did not receive hu-cytokines (left panel). d Myeloid lineage cells after administration of hu-cytokines. CD33⁺, CD15⁺, CD11b⁺, and CD14⁺ cells are also seen in circulating blood after week 12 of CD34⁺ cells administration and when mice (n = 10) receive AAV8 hu-cytokines (see above) plus DNA-hu-cytokines (SCF, FLT-3, THPO; 50 μg; i.m.; multiple sites). An independent batch of mice was used for this experiment. e Reconstituted mice show the presence of CD56⁺ NK (innate immune) cells. Mice (n = 16) bled at 10 weeks showed increased CD56⁺ cells that decrease significantly to physiological levels by week 16 (p = 0.002). An independent batch of mice was used for this experiment. From d, all mice received AAV8 or DNA plasmid-encoded human cytokines as described in the method section. f–i Repopulation of human T- and B-cells. Generally, by 12–14 weeks, physiological levels of human T-and B-cells (f, left panel) and human CD4⁺ and CD8⁺ T cells are observed in circulating blood (f, right panel). Each point in the scatter plot represents blood drawn from an individual mouse (n = 16). g–i Repopulation of lymphoid organs with human immune cells. In H&E staining, there is dense repopulation of human lymphocytes in reconstituted mouse thymus (g; right panel; scale bar: 200 μm) and spleen (h; right panel; scale bar: 250 μm) when compared to non-reconstituted mouse thymus (g; left panel; scale bar: 500 μm) and spleen (h; left panel; scale bar: 200 μm). Dense repopulation of human lymphocytes in mouse kidney (renal capsule) grafted with human thymus (I; scale bar: 200 μm). A representative example of staining is shown in this figure. All observations (g–i) were consistent, and the experiment was repeated more than 2×. All mice were euthanized by CO₂ inhalation/cervical dislocation and organs harvested 24 weeks after CD34⁺ cell injections. Data are presented as mean values ± SD for b–f. A one-sided paired t-test was used for statistical analysis when p-values are specified. Source data are provided as a Source Data file.