Article Figures & Data
Figures
- Figure 1.
Expression of bimodal imaging transgenes using lentiviral vectors. A, A self-inactivating lentiviral system based on HIV-1 (CS-CGW) was used to construct the following vectors: fusion between GFP and Fluc, GFP and Rluc, Fluc and DsRed2, and Rluc and DsRed2 under the CMV promoter. B, hNSCs and glioma cells were transduced in culture with these lentiviral vectors at MOI = 1 and visualized for GFP or DsRed2 fluorescence. Magnification, 20×.
- Figure 2.
Characterization of transduced hNSCs. A, B, hNSCs were transduced with LV-GFP-Fluc, and growth of transduced and nontransduced cells in culture was compared over 15 d (A); immunohistochemistry was performed with anti-nestin antibody and detected with Alexa-540-conjugated secondary antibody (B). C, D, hNSCs or LV-Lacz transduced hNSCs were differentiated for 7 d, and immunocytochemistry was performed with βIII-tubulin and GFAP antibodies and detected with CY3- or CY5-conjugated antibody. Cell nuclei were counterstained with Hoescht 33258. E, Percentage of βIII-tubulin+ cells and GFAP+ cells. Data represent means ± SEM (n = 4). Magnification: B, 20×; C, D, 40×.
- Figure 3.
Bioluminescence characteristics of hNSCs. Different concentrations of either NSCs transduced with LV-GFP-Fluc or LV-Rluc-DsRed2 were either plated or implanted intraparenchymally in the mouse brain. A, B, Transduced hNSCs were plated in concentrations ranging from 2 × 104 to 1.5 × 105, and 12 h later, cells were incubated with 150 μg/ml d-luciferin or 1 μg/ml coelenterazine and imaged under the CCD with a scan time of 1 min. C, D, Mice bearing transduced hNSCs were injected either with 4.5 mg/mouse of d-luciferin intraperitoneally or 100 μg/mouse of coelenterazine and imaged under the CCD with a scan time of 5 min. The photon intensities from GFP-Rluc and Rluc-DsRed2-expressing hNSCs both in vitro and in vivo are plotted.
- Figure 4.
Bioluminescence imaging of NSCs in mice brains. A, hNSCs or mNSCs transduced with LV-GFP-Fluc were plated and 24 h later imaged for bioluminescence. The photon intensities from mNSCs and hNSCs expressing GFP-Fluc are plotted as percentage relative to hNSCs. B, hNSCs or mNSCs expressing GFP-Fluc were implanted intraparenchymally in nude and SCID mice and followed by Fluc bioluminescence imaging on day 3, 7, and 10 d of implantation. One representative image of each mice group on day 10 is shown. The photon intensities from mNSCs and hNSCs expressing GFP-Fluc are plotted as percentage survival relative to mNSCs in nude mice at 3 d (*p < 0.05 vs mNSCs nude).
- Figure 5.
hNSC survival in mice bearing gliomas. hNSCs expressing GFP-Fluc alone or mixed with Gli36 glioma cells expressing Rluc-DsRed2 were implanted intraparenchymally in SCID mice, and the presence of hNSCs and glioma cells was imaged by dual bioluminescence imaging. A, Fluc imaging showing the presence of hNSCs and Rluc imaging showing the presence of glioma cells. B, Fluc bioluminescence intensities of hNSCs expressing GFP-Fluc over time. One representative image of mice with hNSCs and with (+) or without glioma cells is shown. The photon intensities are plotted as percentage hNSC survival (*p < 0.05 vs hNSCs). C, Rluc bioluminescence intensities of glioma cells expressing Rluc-DsRed2 implanted with and without hNSCs and plotted as percentage glioma volumes over time. D, Mice brains implanted with hNSCs expressing GFP-Fluc alone were sectioned, and confocal microscopy was performed. Photomicrographs of brain sections at the implantation depth (i.e., 2 mm) (a, c, e) and 3 mm (b, d, f) are shown. Magnification: D, 10×.
- Figure 6.
hNSCs migrate into gliomas in vivo. A–D, Bioluminescence imaging of mice implanted with GFP-Fluc-expressing hNSCs in mice with established Rluc-DsRed2 gliomas. Fluc images of mice on day 3 (A), day 7 (B), and day 10 (C) and Rluc image on day 10 (D) are shown. E–G, Intravital microscopy of Fluc-DsRed2 hNSCs implanted in mice with established GFP-Rluc gliomas on days 3 (E), 7 (F), and 10 (G) after hNSC implantation. Magnification, 30×. H, Summary data of hNSC cell density at the site of hNSC implantation and colocalized with established glioma at 3, 7, and 10 d after implantation (*p < 0.05 vs hNSCs at implantation site). I–K, Intravital microscopy of Fluc-DsRed2 hNSCs implanted in non-tumor-bearing mice on days 3 (I), 7 (J), and 10 (K) after hNSC implantation.
- Figure 7.
hNSCs are present specifically in the gliomas in an undifferentiated state. Mice implanted with Gli36-GFP-Rluc glioma cells stereotactically into the right frontal lobe were implanted with Fluc-DsRed2 hNSCs 2 d later. Mice were imaged by intravital microscopy (see Fig. 6) and killed on day 10 after hNSC implantation. Mice brains were sectioned, and confocal microscopy was performed. A, Light image showing the normal brain (NB) and the tumor (T). B, Fluorescent image of A showing hNSCs (red) infiltrating the tumor (green); 10× magnification. C, Higher magnification (40×) image of B. D, Brain sections were stained with 1 μm TO-PRO-3 (nucleic acid staining) and confocal microscopy was performed. D, TO-PRO staining (blue). E, GFP-positive glioma cells. F, DsRed2 hNSCs. G, Merge of D–F. H–S, Immunohistochemistry on brain sections from Gli36-glioma-bearing mice implanted with hNSCs expressing GFP-Fluc, 10 d after implantation. Representative images of brain sections immunostained for nestin (H, L, P), Ki67 (I, M, Q), GFAP (J, N, R), and MAP-2 (K, O, S) are shown. Green, GFP expression; red, Ki67, GFAP, or MAP-2 expression; yellow, coexpression of GFP and nestin.
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