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. 2013 Aug 29;4(8):e779.
doi: 10.1038/cddis.2013.304.

Mesenchymal stromal-cell transplants induce oligodendrocyte progenitor migration and remyelination in a chronic demyelination model

J Jaramillo-Merchán  1 , J Jones, J L Ivorra, D Pastor, M C Viso-León, J A Armengól, M D Moltó, E Geijo-Barrientos, S Martínez
Affiliations

Mesenchymal stromal-cell transplants induce oligodendrocyte progenitor migration and remyelination in a chronic demyelination model

J Jaramillo-Merchán et al. Cell Death Dis. .

Abstract

Demyelinating disorders such as leukodystrophies and multiple sclerosis are neurodegenerative diseases characterized by the progressive loss of myelin that may lead toward a chronic demyelination of the brain's white matter, impairing normal axonal conduction velocity and ultimately causing neurodegeneration. Current treatments modifying the pathological mechanisms are capable of ameliorating the disease; however, frequently, these therapies are not sufficient to repress the progressive demyelination into a chronic condition and permanent loss of function. To this end, we analyzed the effect that bone marrow-derived mesenchymal stromal cell (BM-MSC) grafts exert in a chronically demyelinated mouse brain. As a result, oligodendrocyte progenitors were recruited surrounding the graft due to the expression of various trophic signals by the grafted MSCs. Although there was no significant reaction in the non-grafted side, in the grafted regions oligodendrocyte progenitors were detected. These progenitors were derived from the nearby tissue as well as from the neurogenic niches, including the subependymal zone and dentate gyrus. Once near the graft site, the cells matured to myelinating oligodendrocytes. Finally, electrophysiological studies demonstrated that axonal conduction velocity was significantly increased in the grafted side of the fimbria. In conclusion, we demonstrate here that in chronic demyelinated white matter, BM-MSC transplantation activates oligodendrocyte progenitors and induces remyelination in the tissue surrounding the stem cell graft.

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Figures

Figure 1
Figure 1
Effect of chronic intake of cuprizone in the CNS. (a) Chronogram of the experimental protocol. (b) Schematic representation of a transveral section of the brain, indicating with red squares the CC (c) and fimbria (d). (c) Immunohistochemistry of a transveral section of the CC of a mouse treated for 3 months with cuprizone and stained for MBP (in green). Arrows indicate patches of demyelination (MBP-negative), similar to that seen in chronic lesions of MS. (d) Proteolipid protein (PLP) (red) and MBP (green) staining in the fimbria of a non-treated mouse and of a cuprizone-treated mouse of the same age. Note the lack of expression of the two myelin-forming proteins, indicating a homogenous demyelination process of the fimbria. Scale bar, 75 μm (c and d)
Figure 2
Figure 2
OPCs around the graft site. (a) Diagram depicting the location of the injection site. The fimbria (Fi) is adjacent to the CC and the hippocampus, subdivided into CA1, CA2 and CA3. (b) MSC-grafted fimbria immunostained for H2d (green) and NG2 (red) in the injection site. In all cases, the graft stimulated OPC clustering. (c) Stem cell-treated fimbria immunostained for GFP (green) and MBP (red) in the injection site. (d) Close-up image of a region of the periphery of the graft (inset in c) staining for MBP (red) and GFP (green) where the stem cells can be seen intertwined with myelinated fibers. (e) NG2, MBP and GFAP expression (in red) in the fimbria of mesenchymal stem cell-grafted mice, sham-operated and the control demyelinated fimbria of the grafted mice. Note that the NG2 in MSC was taken 1 mm rostrally from the graft where no MSC were present. (f–h) Quantification of the expression of NG2 (f), MBP (g) and GFAP (h) in the fimbria of MSC-grafted mice (green bars), sham-operated (yellow bars) and the control fimbria (blue bars) of both groups. A significant increase of the NG2 expression (t=−3.19; P-value=0.013), MBP expression (t=−4.34; P-value=0.004) and GFAP expression (t=−2.60; P-value=0.032) was detected in the MSC-grafted mice compared with the sham-operated mice, whereas no significant differences were detected in both control demyelinated fimbria (corresponding to the contralateral side of the fimbria) in sham-operated and control demyelinated fimbria in MSC-grafted. Scale bar, 100 μm
Figure 3
Figure 3
Axon conduction velocity in various regions of the fimbria. (a and b) Images of the extracted fimbria, depicting the location of the graft. (c) The stimulus electrode located in the caudal region (c) of the fimbria and a registry electrode in the rostral area (caudal to the graft) (r). (d) Details of the position of the two registry electrodes to obtain the recordings of the caudal fibers around the graft (white line). (e and f) Two fibers were detected: fast (thicker myelin sheaths) and slow (thin and/or non-myelin sheaths) fibers. (g) Histogram depicting the axon potential velocities in the fast fibers measured in the MSC (green), normal non-demyelinated (black), sham-operated (yellow) and control demyelinated fimbria (blue) in the rostral (R) or caudal (C) regions. Red line denotes wild-type values. Scale bar, 1 mm (a and b); 500 μm (c); and 100 μm (d)
Figure 4
Figure 4
Trophic factor expression after grafting. (a) PCR analysis of cultured mesenchymals stromal cells. (b) Immunohistochemical analysis of the stem cell-treated fimbria for the expression of various trophic factors compared with the control fimbria. NT4/5, NT3, PDGF and NGF-β were expressed in MSC-grafted fimbria. The histogram to the right depicts the quantification of the immunoreactivity of the trophic factors analyzed by immunohistochemistry, relative to the contralateral (non-treated) side of the fimbria. A value over 1 indicates a higher immunoreactivity in the treated side of the fimbria compared with the non-treated side. Scale bar, 50 μm
Figure 5
Figure 5
Analysis of the neurogenic niches: subventricular zone. (a–f) Coronary sections of the LV showing NG2+ cells migrating from the subventricular zone to the MSC-grafted fimbria. NG2+ cells, corresponding to OPCs, migrate rostro-caudally from the subventricular zone in the side of the fimbria where the stem cells were injected (a). The cells migrated bordering the lateral ventricular until they reached the fimbria (b–f). (b) Immunohistochemical image depicting the NG2+ cells bordering the LV. (c) Close-up of inset in b, where the cells can be seen with a migratory morphology. (d) NG2+ cells entering the fimbria. (e) Close-up of the inset in d. (f) NG2+ cells surrounding the stem cell graft. The scheme to the right shows the spatial distribution of the images observed in a–f. g, graft; Fi; fimbria; lv, lateral ventricle; Hi, hippocampus. (g) Immunohistochemical analysis of the migrating NG2+/Nilo1+ cells. In the image, H2d is in green (Alexa Fluor 488), NG2 in red (Texas Red), Nilo1 is light blue (aminomethyl coumarin acetate) and 4′,6-diamidino-2-phenylindole in dark blue. Double-positive NG2 and Nilo1 cells are detected in purple. (h–i) The contralateral side of the fimbria, where no cells were injected, did not present the migrating cells observed in the treated side. Scale bar, 100 μm (c and h); 75 μm (e); 50 μm (a, b, d, f and i); and 25 μm (g)
Figure 6
Figure 6
Analysis of neurogenic niches: DG. (a and b) Horizontal sections of the contralateral (non-treated) and MSC-grafted side of the fimbria, respectively, showing the tight location of the DG and fimbria, delimited by yellow-dotted lines. Blue is 4′,6-diamidino-2-phenylindole staining. (c and d) MBP staining, in red, of the rostral (c) (see ‘r' yellow line in the localization scheme) and caudal (d) regions (see ‘c' yellow line in the localization scheme) to the graft (see ‘g' green line in the localization scheme), showing that the area closest to the DG expresses more MBP. (e) Reconstructed semi-thin ultramicrotome sagittal section showing cells migrating from the DG to the fimbria. (f–h) High magnifications of insets in e. Red arrows in f and g indicate cell polarity according to a possible cell migration from the DG. Red arrowheads in h indicates cytoplasmic prolongations from a oligodendrocyte ensheathing various axons. (i and j) Oligodendrocyte precursors migrating toward the graft, as observed by electron microscopy. Scale bar, 100 μm (a and b); 50 μm (c–e); 10 μm (f and g); and 5 μm (h)
Figure 7
Figure 7
Schematic review of bone marrow-derived MSC-induced remyelination. In the top image, the stem cells (labeled G) were grafted into the fimbria (Fi), attracting oligodendrocyte progenitors from the hippocampus (Hi). R is rostral and C is caudal. The bottom image depicts a scheme representing the migration of oligodendrocyte progenitors from the two neurogenic niches: the SEZ (red circles) and subgranular zone (SGZ) of the DG (green circles)
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