Additional file 1: Figure S1. of Intraspinal bone-marrow cell therapy at pre- and symptomatic phases in a mouse model of amyotrophic lateral sclerosis

Quantification of neurons in the anterior horn of the spinal cord. The number of interneurons (NeuN-positive cells with cross-sectional area ≤250 μm2) was analyzed in week 12 in the presymptomatic injected animals a and in week 15 in the symptomatic injected animals b. There was no difference in the number of interneurons in the SOD1G93A mice compared with the wild-type animals. The number of motor neurons was analyzed in the end stage of the disease in the presymptomatic injected animals c and in the symptomatic injected animals d. Both saline-injected and BMMC-injected mice showed a decrease in the number of motor neurons compared with the wild-type mice at the time point analyzed. ***p <0.001. (TIF 745 kb

Distribution of Mesenchymal Stem Cells and Effects on Neuronal Survival and Axon Regeneration after Optic Nerve Crush and Cell Therapy

<div><p>Bone marrow-derived cells have been used in different animal models of neurological diseases. We investigated the therapeutic potential of mesenchymal stem cells (MSC) injected into the vitreous body in a model of optic nerve injury. Adult (3–5 months old) Lister Hooded rats underwent unilateral optic nerve crush followed by injection of MSC or the vehicle into the vitreous body. Before they were injected, MSC were labeled with a fluorescent dye or with superparamagnetic iron oxide nanoparticles, which allowed us to track the cells <i>in vivo</i> by magnetic resonance imaging. Sixteen and 28 days after injury, the survival of retinal ganglion cells was evaluated by assessing the number of Tuj1- or Brn3a-positive cells in flat-mounted retinas, and optic nerve regeneration was investigated after anterograde labeling of the optic axons with cholera toxin B conjugated to Alexa 488. Transplanted MSC remained in the vitreous body and were found in the eye for several weeks. Cell therapy significantly increased the number of Tuj1- and Brn3a-positive cells in the retina and the number of axons distal to the crush site at 16 and 28 days after optic nerve crush, although the RGC number decreased over time. MSC therapy was associated with an increase in the FGF-2 expression in the retinal ganglion cells layer, suggesting a beneficial outcome mediated by trophic factors. Interleukin-1β expression was also increased by MSC transplantation. In summary, MSC protected RGC and stimulated axon regeneration after optic nerve crush. The long period when the transplanted cells remained in the eye may account for the effect observed. However, further studies are needed to overcome eventually undesirable consequences of MSC transplantation and to potentiate the beneficial ones in order to sustain the neuroprotective effect overtime.</p></div

MSC transplantation increased RGC survival 16 days after optic nerve crush.

<p>A-F: Confocal images of flat-mounted retinas labeled with Tuj1 (A-C) or anti-Brn3a (D-F) antibodies. Insets in A-C are higher magnifications of the images to illustrate the morphology of the Tuj1-positive cells. In the control retina (contralateral eye) numerous cells are labeled with Tuj1 (A) and Brn3a (D), and axon bundles are intact (arrows in A). Sixteen days after optic nerve crush, there was a large reduction in the number of RGCs (B and E) and axon bundles were notably compromised, as revealed by Tuj1 staining (arrows in B). In the MSC-injected animals (C and F) the number of surviving RGCs was increased compared to the vehicle-injected ones, and Tuj1 staining in axon bundles was similar to the control (arrows in C). G-J: Quantification of RGC survival 16 days after nerve crush using Tuj1 (G, H) or Brn3a labeling (I, J). Results are displayed as mean ± SEM of the percentage of Tuj1+ or Brn3a+ cells relative to the control retina (contralateral eye). *P<0.05; **P<0.01; ***P<0.001. Scale bar in A-F: 50 µm.</p

MSC transplantation increased RGC axon regeneration 16 and 28 days after optic nerve crush.

<p>A-D: Photomontage of confocal images of optic nerve sections to illustrate axonal outgrowth. CTB-488 was injected into the vitreous body 2 days prior to euthanasia to label the axons. Sixteen days after nerve crush (A,B,E) only a few axons had crossed the lesion site (asterisk) in the vehicle-injected animals (A) and this number was larger in the MSC-injected animals (B). Twenty-eight days after optic nerve injury (C,D,F), the MSC-injected group had an even larger number of axons regenerating beyond the crush site, compared to the vehicle-injected group. E, F: Quantification of CTB-488⁺ axons per nerve at different distances from the crush site (from 0.25 to 2.0 mm). Results are displayed as mean ± SEM. *P<0.05; **P<0.01. Scale bar: 200 µm.</p

MSC transplantation increased RGC survival 28 days after optic nerve crush.

<p>A-C: Confocal images of flat-mounted retinas labeled with Tuj1 antibody (A-C). D-F: Photomontage of confocal images of flat-mounted retinas labeled with Brn3a antibody. A, D: Control retina (contralateral eye). B, E: Twenty-eight days after optic nerve injury, there was a large reduction in the number of RGCs, and axon bundles were thinner (arrows in B) in the vehicle-treated group compared to control. C, F: In MSC-injected animals, the number of surviving RGCs increased compared to vehicle-injected animals, and axon bundles were similar to control (arrows in C). G-J: Quantification of RGC survival 28 days after nerve crush at 1.0 mm or 3.5 mm from the optic disc, using Tuj1 (G, H) or Brn3a labeling (I, J). Results are displayed as mean ± SEM of the percentage of Tuj1+ or Brn3a+ cells relative to the control retina (contralateral ye). *P<0.05; **P<0.01; ***P<0.001. Scale bar: 50 µm (A-C); 500 µm (D-F).</p

MSC transplantation increases FGF-2 and IL-1β expression in the retinal ganglion cell layer.

<p>(A-H) Confocal images of FGF-2 and IL-1β expression in retinas from vehicle injected (A-D) and MSC treated (E-H) groups. TOPRO (C, G) was used for nuclei staining. Images are representative of 3 animals per experimental condition. Scale Bar: 20 µm. (I, J) Graphs show the average mean gray value of Z stack images normalized by the vehicle injected group. *p<0.05, unpaired t-test. RGCL: retinal ganglion cell layer. Scale bar: 50 µm.</p

MSC immunophenotyping by flow cytometry.

<p>MSC were analyzed from passages 3 to 5, after being frozen and thawed. (A) Forward Scatter (FSC) and Side Scatter (SC) profiles of MSC. The gate in (A) was drawn to exclude doublets of cells. Ninety-five percent of the analyzed cells were positive for CD90 and CD29 (B), 99.8% were negative for CD45 and CD11b-c (C), 99.8% were negative for CD34, and 99.6% were negative for CD45 (D). These results are consistent with the phenotype of multipotent mesenchymal stem cells.</p

Detection of FeraTrack-labeled MSCs by <i>in vivo</i> MRI.

<p>MSC were labeled with Fera Track for 4 h and injected in the vitreous body of the left eye after optic nerve crush. (A-L) Representative images of <i>in vivo</i> MRI at coronal (A,E,I), horizontal (B,F,J), and sagittal planes (C,G,K; at left eye level and D,H,L; at right eye level) at different survival times. Arrows indicate hypointense (black) spots corresponding to FeraTrack-labeled cells in the vitreous body of the left eye. Labeled cells were found at 2 (A-C), 14 (E-G) and 18 weeks (I-K) after optic nerve crush and cell transplantation. The right eye was injected with the washing solution of the cells; no signal was detected (D,H,L and right hemisphere of coronal and horizontal images). The lenses are circled with dashed lines; right (R) and left (L) hemispheres.</p

Anterograde labeling with CTB-488.

<p>(A) Photomontage of images of the superior colliculus of a rat 2 days after intravitreous injection of CTB-488 (green). In the absence of optic nerve injury, the tracer is transported from the left eye through the optic nerve until it reaches the right superior colliculus. (B) Photomontage of images of a nerve 4 days after injury. CTB-488 positive axons (green) do not extend farther into the crush site; B′ shows higher magnification. Nuclei (blue) were stained with DAPI. Scale bars: 250 (A), 1000 (B) and 100 (B′) µm.</p