Transgene transfer and GFP expression after transduction of human CD34+ and Jurkat cells.

A Human CD34⁺ blood cells from umbilical cord blood and Jurkat cells were transduced by spin inoculation with lentiviral vectors encoding a nuclear variant of the eGFP at multiplicity of infection (MOI) of 30 and 120 and cultured in vitro. After 5 days, cells were analyzed for GFP expression by both flow cytometry and fluorescence microscopy. B Estimates of the efficiency of transduction based on the percentage of GFP⁺ cells. C Percentage of cells expressing CD34 after 5 days of culture. D, E A representative experiment showing GFP expression after transduction of human CD34⁺ or Jurkat cells at the MOI of 30. In (E), results are represented as histograms of GFP fluorescence intensity (x-axis, log scale) versus cell number (y-axis, linear).

Transplantation of human JK-GFP cells in zebrafish embryos, colonization of hematopoietic organs and proliferation.

A Transplantation of JK-GFP cells in the caudal vein of a wildtype zebrafish embryo at 36 h.p.f. B–D Colonization of zebrafish hematopoietic tissues followed from 1 to 7 d.p.T. Colonization of the CHT (B, E, arrows), thymus (F, arrows), fetal liver (G, arrows), kidney (H, arrows). See Video 1. I Live imaging of JK-GFP cells circulation (asterisk) and proliferation (arrow) in the CHT (Time code 00:15 and 00:24). See Video 2. CHT: Caudal Hematopoietic Tissue. Scale bars: (A–D): 250 µm; (E–H):100 µm, (I): 25 µm. Time code in minutes.

Human JK-GFP cells phagocytosis by zebrafish embryonic macrophages.

A Live imaging of JK-GFP cells injected in a zebrafish embryo at 30 h.p.f. (arrows indicate two cells). Starting from 33 min post transplantation, some green cells appear fragmented (C, D arrows). D Inset shows a high magnification of fragmented cells at 61 min (arrows). Each frame is a maximum projection of 3 planes apart 1 μm. See also Video 4. E–J Macrophages recruitment in a 4-d.p.f. Tg (mpeg1:mCherry) zebrafish embryo after injection with PBS (E–G) or with JK-GFP cells (H–J) (arrow). Merged picture (J) shows the accumulation of macrophages around JK-GFP cells. K Live imaging of mCherry-macrophages in a 4-d.p.f. embryo, 2 days after transplantation of JK-GFP cells. High magnification reveals the phagocytosis of green human cells engulfed by red host macrophages. Each frame is a maximum projection of 5 planes apart 0.8 μm.See also Video 5. Scale bars: (A–D): 25 µm, (E–J): 250 µm, (K): 20 µm. Time code in minutes.

CD34-GFP cells phagocytosis by zebrafish embryonic macrophages.

A, B Z-stack confocal imaging of mCherry-expressing macrophage behavior in a zebrafish embryo 2 h post transplantation (h.p.T.) of CD34-GFP, reveals the accumulation of macrophages around green cells and their phagocytosis (inset C, D and E). A Bright field imaging of caudal hematopoietic tissue in the tail region at 30 h.p.f., merged with confocal fluorescent imaging of CD34-GFP cells and mCherry-expressing macrophages (B). C–E” 3 successive z-stacks confocal planes apart 0.8 μm of phagocytosis of CD34-GFP cells by mCherry-expressing macrophage (arrows). See also Video 6. A aorta, CV caudal vein, NC notochord. Scale bars: A, B: 25 µm; C–E”:10 µm.

Comparison of injection sites in the zebrafish embryo.

A Different injection sites of zebrafish larvae used in this study. Injection of JK-GFP cells in swim bladder (B, C), hindbrain ventricle (D, E) and yolk sac (F, G), of 30 h.p.f. Tg(mpeg1:mCherry) zebrafish embryos. Merged pictures in the insets show macrophages accumulation at 6 h.p.T. in the swim bladder (C) and the hindbrain ventricle (E) but not in the yolk sac (G). Arrows indicate the site of injection. AGM aorta-gonad-mesonephros, CHT caudal hematopoietic tissue. Scale bar: 250 µm.

Genetic and chemical depletion of macrophages in zebrafish embryos.

Primitive macrophages were genetically and chemically depleted before cell engraftment in Tg (mpeg1:mCherry) zebrafish embryos. A–D Complete and efficient depletion of macrophages is observed in PU.1 morphant (B) and in Lipo-C-treated (D) embryos at 35 h.p.f and 48 h.p.f, respectively, compared to controls (A and C) which contain fluorescent macrophages (head arrows). E JK-GFP cells injected in PU.1 treated and non-treated zebrafish embryos are followed for 6 days. Injection into the Duct of Cuvier (DC) of MO-buffer-treated embryos. Injection into the Duct of Cuvier (DC) of PU.1 Morphant embryos. F, G Transplantations of JK-GFP (F) and CD34-GFP (G) cells in the Duct of Cuvier (DC) of Lipo-C-treated embryos show a percentage of engraftment comparable to that obtained with injections into the yolk sac. Scale bar: 250 µm. Injection in the yolk sac of PBS-treated embryos. Injection into the Duct of Cuvier (DC) of PBS-treated embryos. Injection into the Duct of Cuvier (DC) of Lipo-C-treated embryos. Data were presented as mean ± SD and are representative of 3 independent experiments with n = 15–20 (E), n = 10–30 (F) and n = 10–30 (G) embryos/group. (ns not significant or p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).