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A Novel Cell-Based Therapy for Contusion Spinal Cord Injury Using GDNF-Delivering NIH3T3 Cells with Dual Reporter Genes Monitored by Molecular Imaging

¹ Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei, Taiwan; ² Department of Nuclear Medicine, Taipei Medical University, Taipei, Taiwan; ³ Graduate Institute of Biomedical Materials and Engineering and Stem Cell Center, Taipei Medical University, Taipei, Taiwan; ⁴ Department of Physiology, College of Medicine, Taipei Medical University, Taipei, Taiwan; ⁵ Graduate Institute of Neuroscience, College of Medicine, Taipei Medical University, Taipei, Taiwan; ⁶ Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan; ⁷ Taipei Municipal Wan Fang Hospital and Taipei Medical University, Taipei, Taiwan; ⁸ Division of General Surgery, Department of Surgery and Cancer Center, Taipei Medical University Hospital, Taipei, Taiwan; and ⁹ Experimental Diagnostic Imaging, M.D. Anderson Cancer Center, Houston, Texas

View larger version (37K): [in this window] [in a new window]   FIGURE 1.  In vivo imaging of migration and targeting of 131I-FIAU-labeled NIH3T3-TG, GDNF-delivering cells in rats. (A) Representative serial planar imaging of SCI-induced rats (n = 6) at 2, 24, and 48 h after transplantation of 1 x 106 NIH3T3-TG cells labeled with 131I-FIAU. Labeled NIH3T3-TG cells could be detected at injury site as early as 2 h after transplantation. (B) Optical eGFP fluorescence imaging of same rat from 48-h planar imaging. Fluorescence imaging demonstrated migratory pattern of transplanted NIH3T3-TG cells similar to one observed by planar imaging. White line and asterisk represent injury site and transplantation site, respectively.

View larger version (89K): [in this window] [in a new window]   FIGURE 2.  Serial histologic examination of transplanted NIH3T3-TG cells in rat contused spinal cord sections. Numerous eGFP-positive cells (arrows) were detected at site of injection (L1) initially at 2 h (A), 48 h (B), and 3 wk (C) after transplantation. Inserts in right panels showed morphology of NIH3T3-TG (arrows) in higher magnifications.

View larger version (83K): [in this window] [in a new window]   FIGURE 3.  Immunohistologic examination of GDNF-expressing cells in SCI-induced rat spinal cord. (A) Representative serial cross-section of injured cord 3 wk after transplantation. Numerous eGFP-containing cells revealed by immunoperoxidase staining were located mainly around injury epicenter (T10, i) and injection site (L1, iii). Adjacent tissue sections probed with anti-GDNF antibody also exhibited similar staining pattern (ii and iv, respectively). (B) Double immunofluorescence study for both anti-eGFP (i and iv) and anti-GDNF (ii and v) of injured cord 3 wk after transplantation. Cells positive for anti-eGFP and anti-GDNF antisera were detected at injury site (i and ii, respectively) and injection site (iv and v, respectively). Double-labeling of eGFP and GDNF signals was demonstrated by merged images at both injury site (T10, iii) and injection site (L1, vi). Magnification 200x.

View larger version (78K): [in this window] [in a new window]   FIGURE 4.  Prevention of cellular death by GDNF-secreting NIH3T3-TG xenograft. (A) Schematic representation demonstrates collection of circumferential cross-sections of injured cord 3 wk after transplantation. (B) Comparative micrographs illustrating immunofluorescent TUNEL reaction for detection of cellular apoptosis surrounding lesion center of sham-injected (untreated group) (i–iv) and NIH3T3-TG-grafted (v–viii) rat spinal cords. Apoptosis (green) was more pronounced in untreated group (ii, iv), compared with NIH3T3-TG-grafted (vi, viii) cord sections. Nuclei were stained with propidium iodide (iii and vii). When quantified by in vivo TUNEL counts, NIH3T3-TG-grafted spinal cord sections exhibited approximately 2.6-fold less cellular death, compared with untreated group. PI = propidium iodide; FITC = fluorescein isothiocyanate.

View larger version (182K): [in this window] [in a new window]   FIGURE 5.  Colocalization of eGFP- and GFAP-positive cells in NIH3T3-TG-treated rat spinal cord. Serial spinal cord cross-sections were probed with anti-eGFP and anti-GFAP antibodies. At injury epicenter (T10), numerous anti-GFAP (A) and anti-eGFP (B) cells were detected; anti-GFAP and anti-eGFP cells were detected at injection site (L1, C and D, respectively) as well. Arrowheads show immunopositive cells. Inserts in A and B represent magnified images of stained areas. Scale bars = 100 µm.

View larger version (94K): [in this window] [in a new window]   FIGURE 6.  In vitro demonstration of neuronal differentiation potential of NIH3T3-TG cells. (A) NIH3T3-TG cells subjected to neuronal differentiation medium demonstrated elongated and branched morphology, resembling early neuronal cells (i, under fluorescence microscope, eGFP), compared with rat PC12 cells (ii, phase contrast image). (B) Immunofluorescence microscopic analysis of NIH3T3-TG cells after neuronal differentiation. NIH3T3-TG cells (green fluorescence, ii) after neuronal differentiation were both anti-nestin-positive (red fluorescence, i). (C) In vitro gene analysis of differentiated NIH3T3-TG cells. Neuronal-induced NIH3T3-TG cells were immunopositive in vitro for both nestin (i) and GDNF (ii) as well as immunopositive at transcriptional level (insert).