1. Volume 13, Issue 3, Pages 598-608 (March 2006)
A clonal cell line from immortalized olfactory ensheathing glia promotes functional recovery in the injured spinal cord 
M. Teresa Moreno-Flores, Elizabeth J. Bradbury, M. Jesús Martín-Bermejo, Marta Agudo, Filip Lim, Érika Pastrana, Jesús Ávila, Javier Díaz-Nido, Stephen B. McMahon, Francisco Wandosell 
Molecular Therapy 
Volume 13, Issue 3, Pages (March 2006)
DOI: /j.ymthe
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
The TEG3 clonal line was obtained by immortalization of primary cultures of olfactory ensheathing glia using the large T antigen of SV40 (T-SV40). Immunostaining for nuclear T-SV40 (green) and the cytoplasmic marker 3-phosphoglycerate dehydrogenase (red) is shown. TEG3 was maintained in different culture media: (A) M10 and (B) ME3 (see Materials and Methods). Note that the TEG3 cells, which are of clonal origin, change from a majority of cells (>90%) exhibiting a flat polygonal, astrocyte-like morphology in M10 (A) to a majority (>70%) with a bipolar, Schwann cell-like morphology in ME3 (B). Bar, 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
The fate of TEG3 clonal line cells in the spinal cord after injury and transplant. TEG3 cells were incubated with BrdU 18–20 h prior to transplant. Immunostaining for (A, B) BrdU and (C) nuclear SV40 large T antigen (T-SV40) is shown in adjacent sagittal sections (B, C) of the lesioned spinal cord corresponding to a representative animal that survived for 4 weeks after transplant. Most of the cells gathered around the site of the injection (A; low-power view of the lesion site) but had lost the expression of T-SV40 in vivo (compare the number of BrdU-positive nuclei in B with the number of T-SV40-positive nuclei in C). (D) Quantification of the number of TEG3 cells in the spinal cord of animals sacrificed at 2 (n = 4), 4 (n = 3), and 10 (n = 3) weeks posttransplantation. Means ± SEM of the number of BrdU-positive nuclei/field (original magnification ×250) are represented in the ordinate versus the time of sacrifice on the abscissa. ***P < 0.005, ANOVA and Tukey post hoc test. Bars: 100 μm, in the insets 50 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Spinal cord sagittal sections showing the fate of TEG3 cells after transplant into the lesioned spinal cord. TEG3 cells were labeled with BrdU prior to transplantation. (A, C–F) Double immunostaining for BrdU (green) and 3-phosphoglycerate dehydrogenase (PGDH, red). (B) Double immunostaining for BrdU (green) and glial fibrillary acidic protein (red). Animals transplanted with TEG3 (A–C, E, and F) or mock-transplanted with vehicle only (DMEM, D) were sacrificed at 4 (A, B; standard fluorescence microscopy) and 10 weeks (C–F; confocal microscopy) post-injury/transplant. Note the nuclear BrdU (green) and cytoplasmic PGDH (red) immunostaining of TEG3 cells after 4 (A) and 10 (C) weeks in vivo and the capacity of TEG3 cells to acquire Schwann cell-like, astrocyte-like (arrowheads and asterisks, respectively, in C, E, and F), and intermediate morphologies (stars and arrows in C and E) in vivo. TEG3 cells (BrdU-positive green nuclei in B) intermingled with reactive astrocytes (red processes in B). Autofluorescence of debris in the lesion site appears yellow in (B). Bar in A (as for B), 25 μm; in C and F (as for E), 30 μm; and in D, 45 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Ascending regenerating axons of the sensory tracts mediated by TEG3 in the spinal cord after dorsal column crush. The left median and sciatic nerves were labeled by injection of cholera toxin B-subunit (CTB). Immunostaining with anti-CTB was performed to reveal the fasciculi cuneatus and gracilis in animals (A–E) transplanted with TEG3 and (F and G) mock-transplanted with vehicle only (DMEM). Animals were sacrificed at 4 (A–C) and 10 (D–G) weeks after lesion/transplant; (B and C) represent pictures of adjacent sagittal sections of the injured spinal cord of an animal transplanted with TEG3. (B, E, and G) Magnified views of the squares in (A, D, and F), respectively. In (D and F) dashed lines represent the limits between lesion site (asterisk) and the adjacent territory. Axons could be seen navigating through the lesion area (A, D, arrowheads in B and E), which contained many TEG3 cells (C). Growth cone-like endings can be observed in B (arrowheads). In mock-transplanted rats (F and G), few if any CTB-stained fibers were observed within the lesion area (asterisk in F). In fact we found axons caudal to the lesion site (see dashed line in F) with growth cone-like endings turning 180° to face away from the lesioned area (asterisks in G). (A–C) Standard microscopy. (D–G) Confocal microscopy. Bars: A, 100 μm; B and C, 25 μm; D and F, 45 μm; E and G, 30 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Descending regenerating axons of the motor corticospinal tract (CST) promoted by TEG3 in the spinal cord after dorsal column crush. (A–F) Spinal cord sagittal sections. We labeled the CST by injection of biotinylated dextran amine (BDA) into the motor cortex. Immunostaining for BDA (green in A–F) and BrdU (red in A and B) is shown. (A and D) Low-power overview around the lesion site in TEG3- and mock-transplanted rats sacrificed at 10 weeks after transplant. Insets show magnified views of BDA-traced fibers of the CST rostral to the lesion site. (B and C) Magnified views of the left and right squares of (A) and (E and F) of those in (D). Inset in (B) corresponds to a higher power view of BDA-traced fibers in the lesion site. In TEG3-transplanted animals is possible to see CST sprouting and growth cone tips in BDA-stained axons (green, arrowheads in B and inset in B) navigating between TEG3 cells (stained red for BrdU in B and inset in B) at the lesion site. Caudal to the lesion (C), numerous BDA-stained fibers can be observed extending away from the lesion site. In a representative mock-transplanted animal (D), we could see BDA-stained fibers approaching the lesion site (D, closer view of the left square in E). However, no labeled fibers (D, closer view of the right square in F) extend away from the lesion site. In the graph (bottom right), the quantification of the number of BDA-labeled axons in TEG3- and mock-transplanted animals (means ± SEM, n = 3 each group) versus the distance from the lesion is shown (1 unit of distance = 500 μm; point 0 is the center of the lesion site; negative and positive numbers represent areas rostral and caudal to the lesion site, respectively).*P < 0.05 and **P < 0.01 (ANOVA and Tukey post hoc test). To assess total ablation of the CST caudal to the lesion site, we performed immunostaining for PKC-γ (a marker of CST) in transverse sections of the lumbar spinal cord in control and operated animals (10 weeks after lesion/transplant). (G) PKC-γ immunoreactivity in the dorsal horn and CST (arrowhead) of control lumbar spinal cord. (H) In all lesioned animals CST PKC-γ immunoreactivity disappeared at the lumbar level (arrowhead). Bars: A, inset in A, C, and D, 100 μm; B, inset in B, and E (as for F), 45 μm; G (as for H), 200 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
Functional recovery induced by injecting TEG3 cells into the injured spinal cord. The animals (6 unoperated, 6 mock-transplanted with DMEM, and 6 TEG3-treated rats) were tested and subsequently sacrificed at 10 weeks for histological analysis. (A and B) In the tape removal task, lesioned rats mock transplanted with vehicle were severely impaired compared to unlesioned control rats, in their ability both to sense and subsequently to remove the adhesive tape, with significantly higher latency scores throughout the testing period. In contrast, lesioned rats transplanted with TEG3 cells made a moderate functional recovery but did not reach control latencies. (C) In the beam task, lesioned rats mock transplanted with vehicle made significantly more foot-slip errors than controls. In contrast, lesioned rats transplanted with TEG3 cells achieved a marked functional recovery in this task and yielded results that did not differ significantly from those of the control unoperated group. Data are means ± SEM (asterisks denote significant difference from unlesioned control rats: *P < 0.05, **P < 0.01, and ***P < 0.001, ANOVA and Tukey post hoc test).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions