The remyelinating potential and in vitro differentiation of MOG-expressing oligodendrocyte precursors isolated from the adult rat CNS.

There is a long-standing controversy as to whether oligodendrocytes may be capable of cell division and thus contribute to remyelination. We recently published evidence that a subpopulation of myelin oligodendrocyte glycoprotein (MOG)-expressing cells in the adult rat spinal cord co-expressed molecules previously considered to be restricted to oligodendrocyte progenitors [G. Li et al. (2002) Brain Pathol., 12, 463-471]. To further investigate the properties of MOG-expressing cells, anti-MOG-immunosorted cells were grown in culture and transplanted into acute demyelinating lesions. The immunosorting protocol yielded a cell preparation in which over 98% of the viable cells showed anti-MOG- and O1-immunoreactivity; 12-15% of the anti-MOG-immunosorted cells co-expressed platelet-derived growth factor alpha receptor (PDGFRalpha) or the A2B5-epitope. When cultured in serum-free medium containing EGF and FGF-2, 15-18% of the anti-MOG-immunosorted cells lost anti-MOG- and O1-immunoreactivity and underwent cell division. On removal of these growth factors, cells differentiated into oligodendrocytes, or astrocytes and Schwann cells when the differentiation medium contained BMPs. Transplantation of anti-MOG-immunosorted cells into areas of acute demyelination immediately after isolation resulted in the generation of remyelinating oligodendrocytes and Schwann cells. Our studies indicate that the adult rat CNS contains a significant number of oligodendrocyte precursors that express MOG and galactocerebroside, molecules previously considered restricted to mature oligodendrocytes. This may explain why myelin-bearing oligodendrocytes were considered capable of generating remyelinating cells. Our study also provides evidence that the adult oligodendrocyte progenitor can be considered as a source of the Schwann cells that remyelinate demyelinated CNS axons following concurrent destruction of oligodendrocytes and astrocytes.

Schwann cell remyelination is not replaced by oligodendrocyte remyelination following ethidium bromide induced demyelination.

Demyelinated CNS axons can be remyelinated by Schwann cells. A recent study concluded that with time Schwann cell remyelination is replaced by oligodendrocyte remyelination [9]. To examine this, the extent of Schwann cell and oligodendrocyte remyelination at 4, 6.5 and 24 weeks was determined for ethidium bromide lesions made in the spinal cords of rats. Although the extent of oligoden-drocyte remyelination increased with time there was no significant change in the amount of Schwann cell remyelination. This indicates that Schwann cell remyelination is stable and is not replaced by oligodendrocyte remyelination.

Modelling large areas of demyelination in the rat reveals the potential and possible limitations of transplanted glial cells for remyelination in the CNS.

Transplantation of myelin-forming glial cells may provide a means of achieving remyelination in situations in which endogenous remyelination fails. For this type of cell therapy to be successful, cells will have to migrate long distances in normal tissue and within areas of demyelination. In this study, 40 Gy of X-irradiation was used to deplete tissue of endogenous oligodendrocyte progenitors (OPCs). By transplanting neonatal OPCs into OPC-depleted tissue, we were able to examine the speed with which neonatal OPCs repopulate OPC-depleted tissue. Using antibodies to NG-2 proteoglycan and in situ hybridisation to detect platelet-derived growth factor alpha-receptor Ralpha (PDGFRalpha) mRNA to visualise OPCs, we were able to show that neonatal OPCs repopulate OPC-depleted normal tissue 3-5 times more rapidly than endogenous OPCs. Transplanted neonatal OPCs restore OPC densities to near-normal values and when demyelinating lesions were made in tissue into which transplanted OPCs had been incorporated 1 month previously, we were able to show that the transplanted cells retain a robust ability to remyelinate axons after their integration into host tissue. In order to model the situation that would exist in a large OPC-depleted area of demyelination such as may occur in humans; we depleted tissue of its endogenous OPC population and placed focal demyelinating lesions at a distance (< or =1 cm) from a source of neonatal OPCs. In this situation, cells would have to repopulate depleted tissue in order to reach the area of demyelination. As the repopulation process would take time, this model allowed us to examine the consequences of delaying the interaction between OPCs and demyelinated axons on remyelination. Using this approach, we have obtained data that suggest that delaying the time of the interaction between OPCs and demyelinated axons restricts the expression of the remyelinating potential of transplanted OPCs.

Remyelination of demyelinated CNS axons by transplanted human schwann cells: the deleterious effect of contaminating fibroblasts.

Areas of demyelination can be remyelinated by transplanting myelin-forming cells. Schwann cells are the naturally remyelinating cells of the peripheral nervous system and have a number of features that may make them attractive for cell implantation therapies in multiple sclerosis, in which spontaneous but limited Schwann cell remyelination has been well documented. Schwann cells can be expanded in vitro, potentially affording the opportunity of autologous transplantation; and they might also be spared the demyelinating process in multiple sclerosis. Although rat, cat, and monkey Schwann cells have been transplanted into rodent demyelinating lesions, the behavior of transplanted human Schwann cells has not been evaluated. In this study we examined the consequences of injecting human Schwann cells into areas of acute demyelination in the spinal cords of adult rats. We found that transplants containing significant fibroblast contamination resulted in deposition of large amounts of collagen and extensive axonal degeneration. However, Schwann cell preparations that had been purified by positive immunoselection using antibodies to human low-affinity nerve growth factor receptor containing less than 10% fibroblasts were associated with remyelination. This result indicates that fibroblast contamination of human Schwann cells represents a greater problem than would have been appreciated from previous studies.

Remyelination of Demyelinated CNS Axons by Transplanted Human Schwann Cells: The Deleterious Effect of Contaminating Fibroblasts.

Areas of demyelination can be remyelinated by transplanting myelin-forming cells. Schwann cells are the naturally remyelinating cells of the peripheral nervous system and have a number of features that may make them attractive for cell implantation therapies in multiple sclerosis, in which spontaneous but limited Schwann cell remyelination has been well documented. Schwann cells can be expanded in vitro, potentially affording the opportunity of autologous transplantation; and they might also be spared the demyelinating process in multiple sclerosis. Although rat, cat, and monkey Schwann cells have been transplanted into rodent demyelinating lesions, the behavior of transplanted human Schwann cells has not been evaluated. In this study we examined the consequences of injecting human Schwann cells into areas of acute demyelination in the spinal cords of adult rats. We found that transplants containing significant fibroblast contamination resulted in deposition of large amounts of collagen and extensive axonal degeneration. However, Schwann cell preparations that had been purified by positive immunoselection using antibodies to human low-affinity nerve growth factor receptor containing less than 10% fibroblasts were associated with remyelination. This result indicates that fibroblast contamination of human Schwann cells represents a greater problem than would have been appreciated from previous studies.

Transplanted glial cells migrate over a greater distance and remyelinate demyelinated lesions more rapidly than endogenous remyelinating cells.

Glial cell transplantation offers a means of remyelinating areas of demyelination in situations where endogenous remyelination fails. How effective such a strategy would be if undertaken in human demyelinating disease is not yet clear since it is very difficult to create large areas of demyelination in adult rodents that would mimic the situation found in a human disease such as multiple sclerosis. When CNS tissue is subjected to 40 Grays of X-irradiation, remyelination is suppressed in the X-irradiated area unless cells migrate into, or are introduced into the X-irradiated area. In the present experiments, by appropriate positioning of lead shielding we have created a "starting gate" from which oligodendrocyte progenitors must depart in order to colonise areas of demyelination. When the starting gate is located at one end of the area of demyelination, endogenous cells fail to colonise throughout an area of demyelination over the ensuing month. In contrast, when transplanted oligodendrocyte precursors are faced with the same situation, the whole area of demyelination is remyelinated over the same period. To determine how far transplanted cells can migrate to areas of demyelination and also to study how quickly the cells can colonise areas of demyelination we injected cells at some distance from areas of demyelination made in X-irradiated tissue. In these experiments, we found that transplanted cells could repopulate up to 9 mm in 2 months compared to 4 mm recorded for endogenous cells (Franklin et al. [1997] J. Neurosci. Res. 50:337-344). These experiments demonstrate that transplanted cells have a far greater ability to colonise areas of demyelination than endogenous cells.

Identification of a human olfactory ensheathing cell that can effect transplant-mediated remyelination of demyelinated CNS axons.

The olfactory ensheathing cell (OEC) has attracted much interest recently because of its potential for transplantation-based therapy of CNS disease. Rat OECs are able to remyelinate demyelinated axons and support regeneration of damaged axons. Although OECs can be grown readily from the rat, a macrosmatic species, it has been uncertain whether it would be similarly straightforward to obtain these cells from the human, a microsmatic species with a relatively poorly developed olfactory system. In this study, we have identified a human OEC which shares many properties with its rat counterpart, including expression of the low-affinity nerve growth factor receptor (L-NGFr) and similar growth factor requirements. Purified populations of human OECs obtained by selection with L-NGFr antibodies have extremely high viability in tissue culture, and are capable of remyelinating persistently demyelinated CNS axons following transplantation into experimentally induced demyelinating lesions in the rat spinal cord. Thus, the human OEC represents an important new cell for the development of transplant therapy of CNS diseases.

Remyelination occurs as extensively but more slowly in old rats compared to young rats following gliotoxin-induced CNS demyelination.

Age is one of the many factors that influence remyelination following CNS demyelination, although it is not clear whether it is the extent or rate of remyelination that is affected. To resolve this issue we have compared remyelination in young and old adult rat CNS following gliotoxin-induced demyelination. Remyelination of areas of ethidium bromide-induced demyelination in the caudal cerebellar peduncle reached completion by 4 weeks in young adult rats (2 months) but was not complete until 9 weeks in old adult rats (9-12 months). We have also shown that remyelination of lysolecithin-induced demyelination in the spinal white matter of old adult rats (18 months) can be extensive, with longer survival times (8 weeks) than have previously been examined. Thus, it is the rate rather than the extent of remyelination that changes in the ageing CNS. These results have important implications for understanding the mechanisms of remyelination, indicating that remyelination need not occur rapidly for it to be extensive. The capacity for the process of remyelination to continue over many weeks must also be borne in mind when assessing remyelination-enhancement strategies either by transplantation or promotion of endogenous mechanisms.

Local recruitment of remyelinating cells in the repair of demyelination in the central nervous system.

The distance over which remyelinating cells within surrounding intact tissue are stimulated to respond to a demyelinating lesion and migrate toward it is unknown. To address this issue we have conducted a series of experiments in which the generation of remyelinating cells in tissue surrounding a spontaneously repairing area of demyelination induced in the adult rat spinal cord is suppressed by exposure to X-irradiation. By regulating the area of X-irradiation relative to the length of the demyelinating lesion within dorsal white matter we have shown that remyelinating cells are not recruited over distances greater than 2 mm into areas of demyelination, implying that most of the remyelinating cells are locally generated. This result indicates that there is only a narrow rim of normal tissue surrounding an area of demyelination from which remyelinating cells can be recruited. The depletion of cells within this rim may account for the poor remyelination associated with large areas of demyelination and following repeated episodes of demyelination. We have also shown that, in contrast to Schwann cells, oligodendrocyte lineage cells recruited into lesions have a limited ability to rapidly repopulate large areas of demyelination. Attempts to enhance remyelination in situations where it fails should therefore focus on increasing the size of the surrounding area from which remyelinating cells can be recruited by augmenting the level of recruitment signal, and preventing premature differentiation of oligodendrocytes so as to maximize their migratory and proliferative potential.

Schwann cell-like myelination following transplantation of an olfactory bulb-ensheathing cell line into areas of demyelination in the adult CNS.

In this study we have transplanted a clonal olfactory bulb-ensheathing cell line into focal areas of the rat spinal cord which contain demyelinated axons but neither oligodendrocytes nor astrocytes. The cell line was created by retroviral incorporation of the temperature-sensitive Tag gene into FACS-sorted 04+ cells from 7-day-old rat pup olfactory bulb. The spinal cord lesions were obtained by injecting small volumes of ethidium bromide into the dorsal white matter of spinal cord previously exposed to 40 Grays of X-irradiation. Many of the axons were remyelinated by PO+ myelin sheaths 21 days after transplantation. Light and electron microscopy revealed cells engaging and myelinating axons in a manner highly reminiscent of Schwann cells within similar lesions. GFAP+ cells were also present within the lesion. This study provides the first in vivo evidence that olfactory bulb-ensheathing cells are able to produce peripheral-type myelin sheaths around axons of the appropriate diameter.

Remyelination in the CNS of the hypothyroid rat.

A number of studies have provided good evidence to indicate a role for thyroid hormone in myelination. Since myelination and remyelination have many shared objectives, and may therefore involve similar mechanisms, we examined whether thyroid hormone may also have a role in remyelination by both Schwann cells and oligodendrocytes of spinal cord axons that had been demyelinated by the injection of ethidium bromide in thyroidectomized rats. Neither the extent of oligodendrocyte remyelination nor the thickness of myelin sheath formed by remyelinating oligodendrocytes was affected by hypothyroidism. However, the extent of remyelination carried out by Schwann cells was decreased in hypothyroid rats compared with normal control animals.