Figure 1.
Identification of mNSCs in vitro.
(A) Dumbbell-shaped mitotic cells were identified 72 hours after plating mesencephalic cells from E14 rats. (B) By 4 days in vitro (DIV), neurospheres had grown in size and detached from the culture substrate. (C) A floating 7 DIV neurosphere. (D) Fluorescence photomicrogragh showing a neurosphere at 7 DIV immunoreactive for the stem cell marker nestin. Scale bar = 100 µm. Green: FITC.
Figure 2.
Identification of fetal DA neurons in vitro.
Fetal DA neurons were isolated from E18 rat ventral midbrain. (A) Phase-contrast photomicrograph showing cells beginning to elaborate neural processes at 4 DIV. (B) With time in culture, neurites grew longer and thicker. (C, D) Immunofluorescence staining at 9 DIV showing TH-immunopositive neurons with a variety of distinct morphologies (unipolar, bipolar, and multipolar). Green: FITC. (E, F) Fluorescence photomicrograph showing that many cells express both the neuronal marker β-III-tubulin and the DA marker TH. Green: FITC; Red: TRITC. (G) DAPI (blue) nuclear counterstaining; (H) Merged image of E, F, and G. Scale bar = 100 µm.
Table 1.
Mean rotation index.
Figure 3.
Identification of mesencephalic neural stem cells overexpressing GDNF (GDNF-mNCSs).
(A) Fluorescence photomicrographs show that all neurospheres were immunoreactive for the NSCs marker nestin. The neurospheres of peGFPN1-GDNF-transfected mNSCs (termed GDNF-mNSCs) were strong immunopositive for GDNF compare to mNSCs transfected with peGFPN1. Green: FITC, Red: TRITC. Scale bar = 50 µm. (B) Prussian blue staining and transmission electron microscopy confirmed SPIO particles in the cytoplasm of mNSCs. (C) GDNF mRNA expression in GDNF-mNSCs compared to mNSCs transfected with peGFPN1 (termed control mNSCs) was determined by RT-PCR. β-actin was used as an internal control. (D) GDNF protein expression in GDNF-mNSCs and control mNSCs was determined by Western blot. Relative protein expression was normalized to β-actin. The data represents the mean ± SD. *P<0.05 vs. control mNSCs. peGFPN1-GDNF-transfected mNSCs (termed GDNF-mNSCs); mNSCs expressing empty vector (termed control mNSCs).
Figure 4.
In vitro differentiation of SPIO, eGFP double-labeled GDNF-mNSCs.
Fluorescence photomicrographs showing SPIO, eGFP double-labeled GDNF-NSCs growing in a differentiation medium for seven days. eGFP-positive cells (A, D, G, J), immunoreactive for the neuronal markers β-III-tubulin (B) and TH (E), the astrocyte marker GFAP (H), and GDNF (K). Images are merged in the last column (C, F, I, L). Scale bar = 100 µm. Green: FITC, Red: TRITC.
Figure 5.
MR images (T2W/FFE) showing the survival of SPIO-labeled mNSCs in the striatum.
Note the high density of SPIO-labeled mNSCs in each group at the site of injection (striatum), but no detectable extrastriatal signal.
Figure 6.
Survival and differentiation of transplanted cells in the striatum.
Representative photomicrograph illustrating that many transplanted cells survived in the striatum. There were more eGFP+ (green), nestin+ (red), nestin+/eGFP+(merged yellow), TH+ (red), and TH+/eGFP+ cells (merged yellow) in the GDNF-mNSCs+DA group than in the other groups. Scale bar = 100 µm. Green: FITC, Red: TRITC.
Table 2.
Number of cells with specific expression phenotypes and DA neuron differentiation rate after cell transplantation into the rat striatum.