Remyelination, axonal sparing, and locomotor recovery following transplantation of glial-committed progenitor cells into the MHV model of multiple sclerosis

Abstract

The behavior and myelinogenic properties of glial cells have been well documented following transplantation into regions of focal experimental demyelination in animal models. However, the ability of glial cell preparations to remyelinate in such models does not necessarily indicate that their transplantation into demyelinated lesions in clinical disease will be successful. One of the precluding factors in this regard is a greater understanding of the environmental conditions that will support transplant-mediated remyelination. In this study, we determined whether the complex and reactive CNS environment of the mouse hepatitis virus (MHV) model of multiple sclerosis (MS) could support transplant-mediated remyelination. Striatal neural precursors derived from postnatal day 1 mice were committed to a glial cell lineage and labeled. Immunohistochemical staining indicated that this population generated >93% glial cells following differentiation in vitro. Transplantation of glial-committed progenitor cells into the T8 spinal cord of MHV-infected mice demonstrating complete hindlimb paralysis resulted in migration of cells up to 12 mm from the implantation site and remyelination of up to 67% of axons. Transplanted-remyelinated animals contained approximately 2x the number of axons within sampled regions of the ventral and lateral columns as compared to non-transplanted animals, suggesting that remyelination is associated with axonal sparing. Furthermore, transplantation resulted in behavioral improvement. This study demonstrates for the first time that transplant-mediated remyelination is possible in the pathogenic environment of the MHV demyelination model and that it is associated with locomotor improvement.

Fig. 1

The differentiation potential of striatal neural precursors can be restricted by culture conditions. (a) Cell cluster after 5 days of growth in non-adherent growth factor containing media, viewed in phase contrast. Cells were grown as free-floating clusters, reaching approximately 200 μm in diameter. (b) Hematoxylin–eosin-stained cell cluster 1 day after transfer to an adherent substrate. Cells spread out from clusters within 6 h of plating, and by 1 day, possess complex morphologies. (c) Multipolar GalC-positive (green) oligodendrocytes were abundant after 7 days of growth on adherent substrate in the absence of growth factors (Hoechst-positive nuclei are blue). (d) GFAP-positive (green) astrocytes were also abundant after 7 days of growth on adherent substrate in the absence of growth factors (Hoechst-positive nuclei are blue). (e) Few NeuN-positive (red) neurons were present (Hoechst-positive nuclei are blue). (f) The majority of cells (85 ± 10.7%) within cultures were BrdU-positive. This field illustrates BrdU-positive (red) GFAP-positive (green) astrocytes. (g) Quantification of cell types after 7 days of growth on adherent substrate in the absence of growth factors indicated that the differentiation protocol yielded 67.4 ± 4.4% oligodendrocytes, 26 ± 7.4% astrocytes, and 6.6 ± 6.2% other cell types, which included NeuN+ neurons, CD11b + microglia, and other Hoechst-positive cells not identified by the immunostains tested. Error bars represent standard deviation. 40× magnification for a and b, 100× for magnification for c, and d, 400× magnification for e, 200× magnification for f.

Fig. 2

Transplanted glial-committed progenitor cells survived and migrated during the 21-day survival period. (a): BrdU-stained transverse section of the spinal cord from a MHV-infected mouse 21 days following cell transplantation, 4 mm cranial to the site of implantation. BrdU-positive cells are abundant within the white matter tracts. Box is magnified in b. (b) BrdU-positive cells are evenly distributed within the lateral white matter tracts. (c) BrdU-stained transverse section of the spinal cord from a non-transplanted, MHV injected mouse 33 days following MHV injection. Note the absence of BrdU-labeled cells. (d) Distribution of BrdU-positive cells cranial and caudal to the site of implantation. Error bars represent standard deviation. 100× magnification for a, 400× magnification for b and c.

Fig. 3

Transplanted glial-committed progenitor cells differentiated into oligodendrocytes during the 21-day survival period. Double immunohistochemical stains for BrdU to identify transplanted cells and APC-CC1 to identify mature oligodendrocytes. (a) BrdU-stained transverse section of the spinal cord from a MHV-infected mouse 21 days following cell transplantation, 1 mm caudal to the site of implantation. (b) APC-CC1 immunostaining of the same section shown in A. (c) Overlay of a and b. Arrows indicate double-labeled BrdU and APC-CC1 cells, illustrating that transplanted cells differentiated into mature oligodendrocytes. Arrowheads indicate BrdU+ CC1− cells, illustrating transplanted cells that differentiated into other cell types. Arrow profiles indicate BrdU−, CC1+ cells, illustrating endogenous oligodendrocytes. 600× magnification.

Fig. 4

Transplantation of glial-committed progenitor cells resulted in remyelination. (a) Toluidine blue-stained transverse section of a spinal cord from a transplanted animal showing the areas from which photographs and counts were taken. (b) Toluidine blue-stained transverse section of spinal cord white matter from an MHV-infected mouse 33 days after induction of disease illustrating the predominance of demyelinated axons (arrow). (c) Toluidine blue-stained transverse section of spinal cord white matter from an MHV-infected mouse 33 days after induction of disease and 21 days after transplantation of glial-committed progenitor cells. Note that the vast majority of axons bear thin myelin sheaths (arrow) characteristic of remyelination. (d) Toluidine blue-stained transverse section of spinal cord white matter from a normal mouse, indicating normal myelin sheath thickness (arrow), for comparison with panel c. (e) Electron photomicrograph of spinal cord white matter from an MHV-infected mouse 33 days after induction of disease illustrating a demyelinated axon (arrow) and a normally myelinated axon (arrowhead). (f) Electron photomicrograph of spinal cord white matter from an MHV-infected mouse 33 days after induction of disease and 21 days after transplantation of glial-committed progenitor cells. Note the thin myelin sheaths (arrow) characteristic of remyelination (for comparison, note the thickness of the normal myelin sheath in e (arrowhead). (g) Toluidine blue-stained transverse section of spinal cord white matter from an MHV-infected mouse 33 days after induction of disease illustrating the predominance of demyelinated axons amongst vacuoles, macrophages, lymphocytes, and an enlarged extracellular space. (h) Toluidine blue-stained transverse section of spinal cord white matter from an MHV-infected mouse 33 days after induction of disease and 21 days after transplantation of glial-committed progenitor cells. Note the higher proportion of remyelinated axons and total axon density as compared to non-transplanted animals (g). 40× magnification for a, 2000X for b–d, 39 000 for e and f, 800× magnification for g and h.

Fig. 5

Transplantation of glial-committed progenitor cells resulted in remyelination and axonal sparing. (a) Demyelinated axons were present in transplanted and non-transplanted animals; their numbers within 15 000 μm² of white matter were not statistically different (P > 0.05). (b) Remyelination extended 8 mm cranial and 6 mm caudal to the implantation site (arrow) in transplanted animals (the extent of tissue examined) and was significantly greater than the degree of remyelination in non-transplanted animals at every point examined (P < 0.01). (c) Throughout this region, 54% to 67% of the total number of axons in the ventral and lateral columns of transplanted animals were remyelinated. Remyelination in non-transplanted animals was significantly less at every point examined (P < 0.01). (d) The total number of axons within 15 000 μm² of white matter in transplanted animals was approximately 2× the total number of axons within similar regions in non-transplanted animals (P < 0.01 for all points), suggesting that remyelination is associated with axonal sparing. Error bars represent standard deviation.

Fig. 6

Transplantation of glial-committed progenitor cells resulted in an improvement in locomotor abilities. Transplanted animals demonstrated a significant improvement (P < 0.01) in locomotor abilities beginning 14 days after transplantation (arrow). From 24 days after induction of disease, transplanted animals walked with a waddling gait and partial hindlimb weakness, whereas non-transplanted animals demonstrated complete hindlimb paralysis. Error bars represent standard deviation.