Fig. 3

Effect of miR-17 modulation on the hematopoietic reconstitution potential of CB CD34⁺ cells in NPG mice. a Effect of miR-17 modulation on repopulation of CB CD34⁺ cells in NOD prkdc^scid Il2rg^null (NPG™) mice. 4.0 × 10⁴ miR-17 overexpression (17/OE), miR-17 knockdown (17/KD), or control (CTRL) CB CD34⁺ cells were injected intravenously into the sublethally irradiated NPG mice (n = 6 per group). The PB of NPG recipients was temporally monitored by analyzing CD45⁺ population every 4 weeks. The mice were sacrificed at 20 weeks after transplantation and the mononuclear cells from bone marrow were analyzed for human cells composed of CD45⁺, CD45⁻CD36⁺ and CD36⁻GPA⁺ cells and CD45⁺CD34⁺ population by flow cytometry. The level of total human cell engraftment is shown in the left panel. p < 0.03 or 0.04, between the NPG mice injected with 17/OE or 17/KD CD34⁺ cells and those injected with CTRL cells (Student’s t-test). The fraction of CD45⁺, erythroid (CD45⁻CD36⁺ and CD36⁻GPA⁺) and CD45⁺CD34⁺ population among the engrafted human cells is shown in the middle panel. The percent of CD45⁺ cells of PB from NPG recipients was shown in the right panel. *p < 0.05, between the NPG mice injected with 17/OE or 17/KD CD34⁺ cells and those injected with CTRL cells (Student’s t-test). The significant difference was analyzed between the mice injected with 17/OE or 17/KD CD34⁺ cells and those injected with CTRL cells. b Flow cytometry analysis of the human CB CD34⁺ cell repopulation in a representative NPG mouse after miR-17 modulation. Fresh CD34⁺ cells served as controls. The MNCs from bone marrow harvested from the engrafted NPG mice were examined by flow cytometry for the assessment of human cells composed of CD45⁺ cells (R1), erythroid cells including CD45⁻CD36⁺ (R2) and CD36⁻GPA⁺ (R3) populations and CD45⁺CD34⁺ cells (R4). c The levels of miR-17 expression in GFP-positive cells from engrafted mice at 20 weeks were tested by real-time PCR. d The bone marrow MNCs containing the different percentage of human cells (lanes 2–5) from the representative engrafted mice were analyzed for human-specific 17α-satellite DNA by PCR. The human-specific 17α-satellite gene was detected when the human cells were over 0.50 % (lanes 3–5) whereas it was indetectable at a percentage of 0.21 % (lane 2). Lane 1, one mouse without transplants; lane 2, one mouse receiving transplants of 17/OE CD34⁺ cells; lane 3, one mouse receiving transplants of 17/KD CD34⁺ cells; lane 4, one mouse receiving transplants of CTRL CD34⁺ cells; lane 5, one mouse receiving transplants of fresh CD34⁺ cells