Transplanted cultured type-1 astrocytes can be used to reconstitute the glia limitans of the CNS: the structure which prevents Schwann cells from myelinating CNS axons

Transplantation of different glial cells into areas of demyelination made in the adult rat spinal cord allows insights into the cell-cell interaction necessary to reconstruct a glial environment around demyelinated axons. Such studies have shown that type-1 astrocytes are central to the exclusion of Schwann cells from areas of glia-free demyelination. However, for these cells to be established in a manner which prevents Schwann cell remyelination of CNS axons, cells of the O-2A lineage are also required. If cultures of isogeneic rat type-1 astrocytes and mouse O-2A cells are transplanted into lesions made in non-immunosuppressed animals. Schwann cell remyelination is limited and extensive oligodendrocyte remyelination is achieved. This paradigm creates a model of immune mediated demyelination in which the immune response is not primarily directed at oligodendrocyte specific epitopes.     

Type 1 astrocytes fail to inhibit Schwann cell remyelination of CNS axons in the absence of cells of the O-2A lineage.

The extent to which Schwann cells are able to remyelinate demyelinated CNS axons is influenced by the presence of astrocytes. In order to study further the nature of astrocyte control of Schwann cell remyelination in the CNS, cultures containing type 1 astrocytes and a small proportion of Schwann cells, but depleted of O-2A lineage cells by exposure to cytosine arabinoside and complement-mediated immunocytolysis, were transplanted into glial-free lesions in adult rat spinal cord in which the host response to demyelinated axons was suppressed by X-irradiation. Following transplantation of these O-2A lineage-depleted cultures into X-irradiated, demyelinating lesions, there was extensive remyelination of demyelinated axons by Schwann cells, a result which contrasted with those obtained from earlier experiments in which O-2A lineage cells were present within the transplant, and/or recruited from host tissue. This experiment shows that the presence of O-2A lineage cells is required in order for transplanted type 1 astrocytes to organise in a manner which inhibits extensive Schwann cell remyelination of CNS axons.     

Remyelination of demyelinated rat axons by transplanted mouse oligodendrocytes.

The injection of the gliotoxic agent ethidium bromide (EB) into spinal white matter produces a CNS lesion in which it is possible to investigate the ability of transplanted glial cells to reconstruct a glial environment around demyelinated axons. This study demonstrates that transplanted mouse glial cells can repopulate EB lesions in rats provided tissue rejection is controlled. In X-irradiated EB lesions in cyclosporin-A-treated rats, mouse oligodendrocytes remyelinated rat axons and, together with mouse astrocytes, re-established a CNS environment. When transplanted into nonirradiated EB lesions in nude rats, mouse glial cells modulated the normal host repair by Schwann cells to remyelination by oligodendrocytes. In both X-irradiated and non-irradiated EB lesions, transplanted mouse glial cells behaved similarly to isogenic rat glial cell transplants (Blakemore and Crang Dev Neurosci, 1988;10:1-10; J Neurocytol, 1989;18:519-528). These findings indicate that the cell-cell interactions involved in reconstruction of a glial environment are common to both mouse and rat.     

The glial scar and central nervous system repair

Damage to the central nervous system (CNS) results in a glial reaction, leading eventually to the formation of a glial scar. In this environment, axon regeneration fails, and remyelination may also be unsuccessful. The glial reaction to injury recruits microglia, oligodendrocyte precursors, meningeal cells, astrocytes and stem cells. Damaged CNS also contains oligodendrocytes and myelin debris. Most of these cell types produce molecules that have been shown to be inhibitory to axon regeneration. Oligodendrocytes produce NI250, myelin-associated glycoprotein (MAG), and tenascin-R, oligodendrocyte precursors produce NG2 DSD-1/phosphacan and versican, astrocytes produce tenascin, brevican, and neurocan, and can be stimulated to produce NG2, meningeal cells produce NG2 and other proteoglycans, and activated microglia produce free radicals, nitric oxide, and arachidonic acid derivatives. Many of these molecules must participate in rendering the damaged CNS inhibitory for axon regeneration. Demyelinated plaques in multiple sclerosis consists mostly of scar-type astrocytes and naked axons. The extent to which the astrocytosis is responsible for blocking remyelination is not established, but astrocytes inhibit the migration of both oligodendrocyte precursors and Schwann cells which must restrict their access to demyelinated axons.     

Myelination, demyelination and re-myelination in the central nervous system

The myelin sheaths that surround axons in the CNS are made and maintained by oligodendrocytes. These glial cells can form variable numbers of myelin segments (internodules): from 1 to 200 so that when one oligodendrocyte is destroyed with preservation of the axon, many internodules can be lost, constituting a demyelinating process. As a consequence of the destruction of myelin and sheath cells a rapid and abundant cell response takes place. The response is made up by resident (microglia) and haematogenous phagocytes which phagocytose myelin and cellular debris leaving the axons demyelinated. Demyelinated axons may either stay demyelinated and clumped together or they may be separated by astrocytic processes, yet they can be remyelinated. The occurrence of remyelination depends upon the intensity and time of exposition to the demyelinating agent. Remyelination in the CNS with complete restoration of conduction may be made by oligodendrocytes or Schwann cells which invade the CNS when astrocytes are destroyed.     

Schwann cell remyelination is restricted to astrocyte-deficient areas after transplantation into demyelinated adult rat brain

The ability to generate large numbers of Schwann cells from a peripheral nerve biopsy makes them potential candidates for the clinical application of cell transplantation to enhance remyelination in human demyelinating disease. Transplant-derived Schwann cell remyelination has previously been demonstrated in the spinal cord but not for demyelinated axons in the brain, a more likely site for initial clinical intervention. We have transplanted Schwann cells from male neonatal rat sciatic nerves into ethidium bromide-induced areas of demyelination in the deep cerebellar white matter of adult female rats. The extent of Schwann cell remyelination 28 days after transplantation was significantly increased in lesions that received direct injections of Schwann cells compared with non-transplanted lesions. Using in situ hybridisation to identify the rat Y chromosome, transplanted male cells were found to co-localise with the P0 immunoreactive area of Schwann cell remyelination. Combined immunohistochemistry and in situ hybridisation confirmed that many remyelinating Schwann cells were transplant-derived. P0 immunoreactivity and transplanted male cells were found in GFAP-negative, astrocyte-free areas. Transplanted Schwann cells were not identified outside of transplanted lesions, nor did they did not contribute to remyelination of a lesion at a distance from the site of transplantation. Our findings indicate that demyelinated axons in the adult brain can be remyelinated by transplanted Schwann cells but that migration and remyelination are restricted to areas from which astrocytes are absent.     

The presence of astrocytes in areas of demyelination influences remyelination following transplantation of oligodendrocyte progenitors

To date, most experiments examining the myelination potential of transplanted cells have been undertaken into either the immature nervous system or into acutely demyelinating lesions. Since these are situations where myelination or remyelination are occurring, such studies provide little information on the likely outcome of introducing myelinogenic cells into area of chronic demyelination. In an attempt to gain a greater understanding of the interaction between astrocytes and oligodendrocyte progenitors in areas of demyelination, we undertook transplantation experiments in which an identical preparation of oligodendrocyte progenitors (OPCs) was (1) transplanted directly into astrocyte-free areas of acute demyelination (3 days after induction), (2) transplanted cranial to similar areas of demyelination (20 days after induction) or (3) transplanted cranial to areas of demyelination (20 days after induction) that had been injected with astrocytes at 3 days to confront OPCs with demyelinated axons in an astrocytic environment. The acute astrocyte-free lesions were remyelinated by oligodendrocytes and Schwann cells while the delayed interaction of OPCs with demyelinating lesions resulted in only oligodendrocyte remyelination, the extent of which was reduced when the area of demyelination contained astrocytes. The results of these experiments illustrate that the introduction of OPCs into an astrocyte-free area of demyelination soon after its induction favours Schwann cell differentiation while the presence of established astrocytes in an area of demyelination has an inhibitory effect on the extent of oligodendrocyte remyelination achieved by OPCs.     

The differentiation of glial cell progenitor populations following transplantation into non-repairing central nervous system glial lesions in adult animals

The non-repairing nature of the locally x-irradiated ethidium bromide (EB)-induced demyelinating white matter lesion has been further validated by showing that injections of two cultures which promote host remyelination of EB lesions in normal tissue do not do so in x-irradiated lesions. The behaviour of an oncogene-immortalized glial cell line and a growth-factor-expanded glial progenitor population have been examined following transplantation into the non-repairing EB lesion. Our studies indicate that the selected gell populations were eacable of establishing glial environments around demyelinated axons. Extensive oligodendrocyte remyelination with little astrocytic presence was observed in lesions transplanted with growth-factor-expanded optic nerve progenitors, while less extensive oligodendrocyte remyelination with the establishment of astrocyte-like cells was found in lesion transplanted with ts A58-SV40T immortalized glial cells. Prolonged expansion of both populations resulted in a loss of differentiation to normal glial phenotypes.     

Glial cell transplanatation and remyelination of the central nervous system

Glial cell transplantation has proved to be a powerful tool in the study of glial cell biology. The extent of myelination achieved by transplanting myelin-producing cells into the CNS of myelin mutants, or into focal demyelinating lesions has raised hope that such a strategy may have therapeutic applications. Oligodendrocytes or Schwann cells could be used for repair. It is likely that the immature stages of the oligodendrocyte lineage have the best phenotypic characteristics for remyelination when transplanted, either as primary cells or as immortalized cells or cell lines. Prior culturing and growth factor treatment provides opportunities to expand cell populations before transplantation as dissociated cell preparations. Cell lines are attractive candidates for transplantation, but the risk of transformation must be monitored. The application of this technique to human myelin disorders may requier proof that migration, division and stable remyelination of axons by the tranplanted cells can occur in the presence of gliosis and inflammation.     

Schwann cell and oligodendrocyte remyelination in lysolecithin-induced lesions in irradiated rat spinal cord

Localised irradiation of adult rat spinal cord was achieved by implanting for 2 weeks a 192Ir pin alongside vertebral segments in the thoraco-lumbar region of the spinal column. Following removal of the implant, lysolecithin (LPC) was injected directly into the dorsal columns in order to induce demyelination in the most intensely irradiated segments of spinal cord. Eight weeks after LPC injection, remyelination was much less extensive in dorsal columns which absorbed more than 40Gy than in LPC lesions in less intensely irradiated spinal cords or in unirradiated animals. No oligodendrocytes, few astrocyte processes and little myelin debris lay among the demyelinated axons. However, capillary vessels were surrounded by astroglial end-feet so that the glial-limiting membrane remained intact in the demyelinated regions. There were some oligodendrocyte remyelinated fibres around the edges of the demyelinated zones but none among the naked axons. Schwann cells, which probably migrated into the lesions from the proximal segments of the dorsal roots, provided some fibres with myelin sheaths. These remyelinated fibres abutted demyelinated axons without an intervening glial limiting membrane or astrocyte process. Oligodendrocytes may fail to migrate into the demyelinated regions because of the scarcity of astrocyte processes. A possible explanation for the limited Schwann cell remyelination may be that the presence of astroglial end-feet around capillaries deprived Schwann cells of ready access to the demyelinated regions.     

Schwann cell remyelination in experimental herpes simplex encephalitis at the trigeminal root entry zone

Inoculation of mice on the cornea with herpes simplex virus, type I, results in demyelination of central nervous system (CNS) axons at the trigeminal root entry zone. This study examined the process of remyelination in this area. Between eight and 15 days after corneal infection, increasing numbers of Schwann cells appeared on the CNS side of the trigeminal root entry zone, where they encircled the demyelinated CNS axons. Remyelination of CNS axons by Schwann cells began between 12 and 15 days and increased during the following weeks. Remodeling of remyelinated internodes continued during the nine weeks of observation. No infectious virus could be cultured 15 days after infection, although latent virus was recovered from the dorsal root ganglia at this time. The disruption of astrocytes on the CNS side of the trigeminal root entry zone during the early stages of infection and the proximity of Schwann cells to the CNS trigeminal root entry zone appear to be important factors affecting CNS remyelination.     

Transplantation of human olfactory ensheathing cells elicits remyelination of demyelinated rat spinal cord

Human olfactory ensheathing cells (OECs) were prepared from adult human olfactory nerves, which were removed during surgery for frontal base tumors, and were transplanted into the demyelinated spinal cord of immunosuppressed adult rats. Extensive remyelination was observed in the lesion site: In situ hybridization using a human DNA probe (COT-1) indicated a similar number of COT-1-positive cells and OEC nuclei within the repaired lesion. The myelination was of a peripheral type with large nuclei and cytoplasmic regions surrounding the axons, characteristic of Schwann cell and OEC remyelination. These results provide evidence that adult human OECs are able to produce Schwann cell-like myelin sheaths around demyelinated axons in the adult mammalian CNS in vivo.     

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.     

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.     

Failure of remyelination in areas of demyelination produced in the spinal cord of old rats

The extent of remyelination was determined one month after injecting 1μl of 1.0% lysolecithin into the dorsal columns of adult rats of three age groups—juvenile, young and old. In the juvenile group (2 months) all axons were remyelinated by either oligodendrocytes or Schwann cells. In the young rats (5 months) nearly all axons were remyelinated. However, in the oldest age group (> 12 months) many axons remained demyelinated and there was a decrease in both oligodendrocyte and Schwann cell remyelination. Also, myelin sheaths formed by oligodendrocytes in the old rats were thinner than those found in the young rats; evidence of impaired Schwann cell remyelination was also seen. The appearance of the lesions in the old animals was variable and many contained myelin debris despite the presence of macrophages within the demyelination area. Although some astrocytes were present in the lesion, many of the demyelinated axons were not separated by astrocyte processes. It is suggested that the failure of remyelination in the old rats following lysolecithin-induced demyelination may be related to sluggish responses of astrocytes and/or macrophages to demyelination. However, a lack of recruitable myelin-forming cells in old animals cannot be excluded.     

Stem cell repair of central nervous system injury

Neural stem cells (NSCs) have great potential as a therapeutic tool for the repair of a number of CNS disorders. NSCs can either be isolated from embryonic and adult brain tissue or be induced from both mouse and human ES cells. These cells proliferate in vitro through many passages without losing their multipotentiality. Following engraftment into the adult CNS, NSCs differentiate mainly into glia, except in neurogenic areas. After engraftment into the injured and diseased CNS, their differentiation is further retarded. In vitro manipulation of NSC fate prior to transplantation and/or modification of the host environment may be necessary to control the terminal lineage of the transplanted cells to obtain functionally significant numbers of neurons. NSCs and a few types of glial precursors have shown the capability to differentiate into oligodendrocytes and to remyeliate the demyelinated axons in the CNS, but the functional extent of remyelination achieved by these transplants is limited. Manipulation of endogenous neural precursors may be an alternative therapy or a complimentary therapy to stem cell transplantation for neurodegenerative disease and CNS injury. However, this at present is challenging and so far has been unsuccessful. Understanding mechanisms of NSC differentiation in the context of the injured CNS will be critical to achieving these therapeutic strategies.     

Extensive oligodendrocyte remyelination following injection of cultured central nervous system cells into demyelinating lesions in adult central nervous system

Following the injection of central nervous system (CNS) cell cultures, prepared from 1-day-old rats and maintained in vitro for 7 days, into irradiated, demyelinating lesions in the spinal cord of adult isologous animals, extensive remyelination of axons by oligodendrocytes was observed. In addition, astrocytes, within the transplanted cell suspension, established normal relationships with oligodendrocytes, axons and other tissue elements, which led to the establishment of large CNS territories throughout the lesions. Outside these CNS domains, Schwann cells, which are present in the transplanted cell suspension, myelinated groups of axons. These observations indicate that the irradiated, ethidium bromide lesion provides an in vivo environment, devoid of the influences of host glia, in which to examine the interactions of transplanted glial cells with demyelinating axons.     

Porcine neural progenitors require commitment to the oligodendrocyte lineage prior to transplantation in order to achieve significant remyelination of demyelinated lesions in the adult CNS

Glial cell transplantation is a potential therapy for human demyelinating disease, though obtaining large numbers of oligodendrocyte precursors from nonrodent species is currently problematic. Culturing of multipotent neural progenitors may provide a solution to this problem, because these cells can be expanded in vitro whilst retaining the ability to differentiate into both neurons and glial cells. In order to investigate the myelinating capability of multipotent neural progenitors, we isolated cells from the porcine subventricular zone, a region rich in neural progenitors, and transplanted them into areas of persistent demyelination in the spinal cord of immunosuppressed rats, created by the injection of ethidium bromide and subsequent exposure to 40 Gy X-irradiation. Porcine SVZ cells were transplanted either within 12 h of isolation or after 7 days in B104-conditioned medium. Freshly isolated cells did not mature into myelinating oligodendrocytes following transplantation and instead remained as clusters of undifferentiated progenitors. However, cells exposed to B104-conditioned medium prior to transplantation were able to effect complete remyelination of the demyelinated axons. This suggests that neural progenitors must be manipulated in vitro for commitment to the oligodendrocyte lineage prior to transplantation if significant remyelination is to be achieved.