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Replacement of Specific Populations of Cells: Glial Cell Transplantation into the Spinal Cord

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Introduction

In recent years an increasing number of results of successful spinal cord transplantation has been reported. Apart from theoretical interest the main aim of these experiments was to find a possible way to improve the consequences of spinal cord injury or neurological disorders affecting the spinal cord. With few exceptions these studies have focused on the survival of neurones, their ability to express specific neurotransmitters and functional improvements achieved by grafting. Glial cells have also been widely used for transplantation into the brain and spinal cord of normal animals and various mutants. A great number of these studies was concerned with the environment provided by glial cells and the putative trophic factors expressed by the grafts. Apart from grafting the two indigenous macroglial cell types of the CNS, the astrocytes and oligodendrocytes, successful attempts have been made to transplant Schwann cells. These experiments showed that glial cells played an important role in the development, regenerative capacity and function of the spinal cord and the possible use of glial cell grafts in demyelinating and degenerative diseases had been suggested. Apart from some mainly theoretical studies concerning the development of glial cells in an alien environment such as the spinal cord, most of the recent investigations revealed several important features of grafted glia, (oligodendrocytes, their precursors and Schwann cells) namely their excessive capability to migrate and to remyelinate dys- or hypomyelinated CNS areas. Astrocytes were shown to migrate long distances along nerve fibres, blood vessels and through the parenchyma, often as far as the full length of the cord. Similar migratory pattern of oligodendrocytes has been reported by Gout et al1 (1988) in a mouse mutant's (shiverer) spinal cord. This migration of astrocytes is of particular importance since grafted glial cells deposited at a central part of the spinal cord may exert their effect throughout the whole cord soon after grafting. Similarly, grafted oligodendrocytes may remyelinate substantially larger areas by spreading throughout a demyelinating cord. Schwann cells in addition to their extensive capability to migrate encourage lesioned and regenerating axons to extend processes and maintain their myelinating activity within the CNS. They are therefore used for grafting when regeneration of lesioned axons is required.

Apart from macroglia, microglial cells, a subpopulation of parenchymal macrophages have recently been introduced into the potential therapeutical arsenal of glial cells. Although their action seems to be entirely different from that of macroglial cell types, their use suggested some success in helping the recovery of the injured cord.

These investigations were aimed to establish a possible use of glial grafts in human diseases, such as in degenerative disorders so as to ameliorate the function of the degenerated areas, improve the regenerative capability of the injured spinal cord and to achieve some remyelination of demyelinating axons. Below the results obtained from grafting various glial cell types into the spinal cord are summarized and their possible therapeutic use is discussed.

Transplantation of Schwann Cells

Spontaneous remyelination after a demyelinating lesion in some diseases involves the presence of myelinating Schwann cells arising from the periphery. Such remyelination by Schwann cells was observed in the periphery of plaques that developed in patients suffering from multiple sclerosis, in brains affected by experimental allergic encephalomyelitis, Theiler's murine encephalomyelitis virus infection or in focal experimental lesions caused by chemical agents, such as lysolecithine or saporin. In these experimental demyelinating lesions Schwann cells first entered the spinal cord via the dorsal root entry zone and the lateral funiculi and readily remyelinated the spinal cord, resulting in functional recovery. Several weeks after remyelination appeared complete by Schwann cells, endogenous oligodendrocytes expelled the Schwann cells and remyelinated the axons previously being myelinated by Schwann cells without lapse in motor function.2 The progressive replacement of Schwann cells by oligodendrocytes was accompanied by invasion of astrocyte into areas myelinated by Schwann cells (fig. 1).

Figure 1. Transverse L5-6 lumbar spinal cord sections at days 75 (A, B, E) and 150 (C, D, F), immunolabeled for astrocytes (GFAP, green), Schwann cells (Schwann/2E, red), and oligodendrocytes (MAB 1580, blue).

Figure 1

Transverse L5-6 lumbar spinal cord sections at days 75 (A, B, E) and 150 (C, D, F), immunolabeled for astrocytes (GFAP, green), Schwann cells (Schwann/2E, red), and oligodendrocytes (MAB 1580, blue). A, B, Day 75: the previously demyelinated area is now (more...)

This observation of spontaneous Schwann cell remyelination raised the question whether Schwann cells could provide an alternative source of cells to repair lesions in demyelinated areas in the spinal cord.3 Such a possibility appeared feasible because remyelination of peripheral nerves by Schwann cells occurs more rapidly than remyelination in the CNS by host oligodendrocytes. This is probably due to the fact that adult oligodendrocytes have a limited migratory and myelinating capacity while mature Schwann cells are able to proliferate and remyelinate. However, this aggressive capacity to remyelinate axons in the CNS can be sometimes disadvantageous to certain extent (for details see later). Another encouraging aspect of grafting Schwann cells into lesioned spinal cord was their capability to provide a favourable environment for regenerating axons possibly because they sequester putative trophic factors as well as extracellular matrix growth promoting molecules.4 Several authors have reported that neurones from the spinal cord or other part of the CNS are able to extend axons for long distances into peripheral nerve grafts and it was thought that regeneration and remyelination within the CNS may be enhanced by providing the adequate PNS-like environment for damaged axons (for details see Chapter 4).

Studies by Blakemore and colleagues have shown that experimentally demyelinated spinal cord fibre tracts can be partially remyelinated by Schwann cells arising from a peripheral nerve juxtaposed or inserted into the dorsal columns.5-8 The autologous nerves need not be placed into the cord, and placing a piece of peripheral nerve into the subarachnoid space8 or onto the demyelinated dorsal columns also has the desired effect of substantial remyelination.6 However, the Schwann cell-induced repair never exceeded more than 50% remyelination of naked axons.8 As the self-repairing process of the spinal cord was suppressed by a high dose of x-ray irradiation it was assumed that all remyelination was due to the graft. Axons remyelinated by Schwann cells could easily be recognised by the signet-ring-like appearance of Schwann cell myelin and the thicker and more compact myelin sheaths. Schwann cells appeared capable to migrate over short distances in response to the appropriate signal, presumably coming from denuded axons. However, this migration was limited and slow and the entry of Schwann cells into the spinal cord was confined to the perivascular spaces which suggested that there might be a significant difference between the extracellular matrices of the CNS and PNS.8 It has also been shown that although NDFβ (Neu Differentiation Factor β) is an effective mitogen for monkey Schwann cells, but its use does not prevent Schwann cells differentiating into myelinating cells.9

Results similar to those obtained by peripheral nerve grafts were obtained by grafts of cultured autologous Schwann cells placed either into chemically demyelinated cords (ethidium bromide,7,10,11 lysolecithin9,12 or diphtheria toxin13) or into myelin-deficient mutants (quaking mouse12). Focal Schwann cell injections into areas of demyelination resulted in limited migration and myelination pattern12-14 and again, the remyelination was closely related to vessels or demyelinated areas which contained astrocytes. The extent of remyelination is related to the number of grafted Schwann cells and the proliferation of Schwann cells through the lesion rather than to their extensive migration. Extensive remyelination was reported only if

  1. transplantation was performed as early as two days after inducing the demyelination14 when the astrocytic complement of the lesioned spinal cord was still present,
  2. Schwann cells were grafted directly into the lesion15 and
  3. grafted Schwann cells were only minimally contaminated with fibroblasts.16

Normally astrocytes prevent the intrusion of Schwann cells into the spinal cord, but astrocytic populations altered by x-ray irradiation are believed to provide a surface for migratory Schwann cells and produce extracellular matrix components. In areas where no astrocytes were present only the perivascular collagen was able to promote the migration of Schwann cells and in the absence of structural extracellular matrix components Schwann cells could not migrate alongside naked axons, but formed clumps without forming myelin.14 On the other hand, unlike astrocytes of an irradiated spinal cord,7 intact astrocytes limit peripheral remyelination by grafted Schwann cells.12,13,17 The interaction between astrocytes and unmyelinating Schwann cells does not seem to be so important as that between astrocytes and grafted oligodendrocytes (see: Transplantation of oligodendrocytes) and aggressively myelinating Schwann cells often displaced astrocytes from the areas of demyelination.7,18 Another aspect of the remyelination process was the competitive relationship between oligodendrocytes and Schwann cells. Schwann cells remyelination is a much quicker process than that produced by oligodendrocytes7,18 though they do not seem to be mutually inhibitory since they can be found together in areas of remyelination.

Following rapid remyelination such as in demyelinated cat spinal cords remyelinated by Schwann cells Blight and Young18 observed a faster recovery of some electrophysiological functions (such as cortical somatosensory evoked potentials, CSEP) than in cords repaired by oligodendrocytes. However, in the long-term, when remyelination was complete there was no electrophysiological difference between oligodendrocyte and Schwann cell-remyelinated cords and the extent of recovery was strictly related to the axon survival. Based on these morphological and physiological findings it became clear that Schwann cells are able of functional remyelination but this does not necessarily mean complete restoration of axonal function.18 When Schwann cells were co-grafted with astrocytes, remyelination following grafting was near complete and the morphological signs were accompanied by the following electrophysiological features:

  1. restoration of conduction through the lesion,
  2. the remyelinated axons showed enhanced impulse recovery to paired-pulse stimulation and
  3. could fire at higher frequencies compared with both demyelinated and control axons (fig. 2).19

Figure 2. A) Schematic showing the dorsal surface of spinal cord with the positions of the stimulating (S) and recording (R) electrodes.

Figure 2

A) Schematic showing the dorsal surface of spinal cord with the positions of the stimulating (S) and recording (R) electrodes. Shaded region indicates the area of demylination or remylination. B) Compound action potentials recorded at 1 mm increments (more...)

Schwann cells in culture are able to maintain their remyelinating capacity so that they are able to myelinate xenogeneic hosts as well.11,12,17,20 However, with time Schwann cells appeared to regain their histocompatibility antigens and when grafted into an in vivo environment the remyelinating cells were rejected by 30 days after transplantation20 or after discontinuing immunosuppression therapy,12,17 leaving behind demyelinated areas. These results indicated that remyelination was solely due to transplanted cells. Electropysiological analysis has shown that human Schwann cells grafted into a demyelinated rat spinal cord induced functional remyelination suggesting that the conduction block has been overcome.19

The pathways and distances taken by migrating Schwann cells were also characterized by various authors.21-24 Immortalized and purified Schwann cells transplanted at distances from the demyelinating lesion of shiverer and normal spinal cords showed extensive migration towards the lesion. Baron-Van Evercooren et al23 labelled Schwann cells with a fluorescent dye whilst Langford and Owens24 used a β-galactosidase gene labelling by infecting Schwann cells with a recombinant retrovirus. Labelled Schwann cells arrived at the site of lesion two-three days after grafting, began to proliferate and expressed the peripheral myelin protein P0.21-23,25 The migrating Schwann cells preferred pathways along the subarachnoid space,24 ependyma, meninges and blood vessels but not the white matter.22,26 Similarly to previous observations, migration in normal white matter never exceeded two-three mm while along other surfaces Schwann cells migrated at a speed of four mm/day as far as eight mm towards the lesion. In the less compact white matter of the shiverer mouse migration was more extensive but in uninjured animals little or no migration was found.22 This finding together with the progressive directional migration of Schwann cells towards and within the lesion suggested that the lesion site triggers the migration of Schwann cells.21,24

Recent studies have shown that unmyelinating Schwann cells introduced into the CNS environment survive poorly and do not migrate unless they can myelinate axons.26 Moreover, migrating Schwann cells do not seem to interact directly with myelin sheaths and oligodendrocytes.27 These negative features of Schwann cells may be explained by multiple mechanisms acting between Schwann cells and the resident cell types of the CNS, such as the regulation of expression of polysialic acid-NCAM and N-cadherin.26

According to these results, grafted Schwann cells are a very potent cell population of the PNS capable of rapid and effective repair of demyelinated areas when transplanted into the CNS. They are able to migrate towards denuded axons and compete with less potent host oligodendrocytes for remyelination. On the other hand, remyelination by Schwann cells in the CNS is not entirely advantageous. Their aggressive penetration of the host tissue results in deteriorating changes of the microenvironment of the lesion site because Schwann cells may displace both host astrocytes and oligodendrocytes. This uncontrolled myelination may, in turn limit their use as potential cells to be grafted into demyelinated foci of CNS.

Transplantation of Olfactory Ensheathing Cells

Olfactory ensheathing cells (OECs) are resident in the olfactory nasal mucosa where they do not form myelin. They have common properties of both Schwann cells and astrocytes (for details see section on transplantation of OECs in chapter 4 and reviews by Ramon-Cueto and Valverde (1995)28 and Bartolomei and Greer (2000)29). The rationale behind OEC transplantation into demyelinated areas of the spinal cord was that

  1. although OECs normally do not produce myelin, they can myelinate axons in vitro30 and
    their astrocyte-like properties may enhance their myelinating capacity following transplantation.

It was assumed that the above features of OECs make them useful candidates for cell therapy to remyelinate demyelinated lesions. The most impressive series of grafting experiments were performed in the laboratories of Robin Franklin and Jeffery Kocsis.

Transplantation of OECs into an experimentally demyelinated rat spinal cord induced a pattern of remyelination very similar to that produced by co-transplantation of astrocytes and Schwann cells, i.e., a single OEC remyelinated a single axonal segment.31,32 Again, the extent of remyelination was similar to that of the co-grafting experiments: OECs remyelinated axons along the whole lesion site without the need of additional cell types, and the grafted cells showed relatively good migrating capabilities.33 The electrophysiological properties of the remyelinated axons revealed that their conduction velocity returned to near normal values and the conduction block was overcome.

In the above experiments it appeared relatively easy to produce an OEC line from a macrosmatic species, such as rat. It was uncertain, whether the same amount of tissue with the same properties as in the case of rodents, can be obtained from humans. Barnett et al (2000)34 and Kato et al (2000)35 have shown that human OECs obtained from olfactory bulbs and nerves can be purified and maintained in culture. The purified OECs were grafted into demyelinated areas of rat spinal cords where whey induced functional remyelination similar to that of rat OECs.

These studies have shown that human OECs may represent a new cell line for the development of transplantation strategy of CNS disorders and exhibit many properties of the rodent OECs including their capacity to form new myelin in the injured spinal cord.

Transplantation of Oligodendrocytes

Introduction

Oligodendrocytes are specific glial cells responsible for providing and maintaining CNS axons with insulating myelin sheaths, thus enabling rapid conduction of action potentials by axons. Accordingly, any harmful influence on the oligodendrocytes or any intrinsic defect may result in an incomplete or imperfect myelination or gradual loss of existing myelin sheaths. Recently it has become evident that axonal degeneration and/or spinal cord injury induces considerable oligodendrocyte apoptosis, thus reducing the number of glial cells that are able to remyelinate the lesion site.36,37 There are well-known demyelinating diseases, such as the different forms of myelitis or multiple sclerosis. As spontaneous remyelination of demyelinated lesions in the mammalian CNS appears unsatisfactory, the studies on oligodendrocyte transplantation are of outstanding importance, not only because of the possible future use of oligodendrocytes to replace missing cells but also because oligodendrocytes together with astrocytes form a complex and well-balanced glial environment of axons and neurones. To restore such an environment by carefully selected proportion of glial cells naturally occurring in the CNS helps to understand the glia-neurone interaction in the CNS. Again, the spinal cord where long fibre tracts are situated on the external surface of the cord provides an excellent model for the study of migration and remyelination of grafted oligodendrocytes.

Even before successful transplantation of oligodendrocytes into the spinal cord was carried out, there was evidence to show that oligodendrocytes and their precursors are able to myelinate naked axons both in vivo and in vitro. In vivo experiments were carried out on hypomyelinated brains of mutants, such as the shiverer mouse. Hypomyelinated axons possess uncompacted myelin sheaths which lack the so-called major dense line due to the absence of a myelin protein of major importance (in the case of shiverer the Myelin Basic Protein, MBP).38 Normal oligodendrocytes transplanted into a hypomyelinated brain of shiverer mice were able to overcome the defective shiverer oligodendrocytes and remyelinate naked axons.39 Nevertheless, electron microscopic investigations revealed that transplanted and host oligodendrocytes competed for unmyelinated axons.40 Similarly, if explants of ARA-C-treated CNS tissue was kept in vitro and was co-cultured with other tissue explants enriched in oligodendrocytes or cultured oligodendrocytes, remarkable remyelination occurred by the “grafted” oligodendrocytes and the explanted tissue became more mature.41,42 Moreover, the explanted oligodendrocytes migrated towards naked axons.42 The question arose whether oligodendrocytes taken from any part of the CNS are able remyelinate explants to the same extent. Interestingly, the myelinating capacity of dissociated oligodendrocytes or optic nerve explants superimposed upon ARA-C-treated cerebellar explants was less than that of organotypic explants or cultured oligodendrocytes.41,43 The remyelination by “grafted” oligodendrocytes proved genotype-specific, i.e., shiverer oligodendrocytes formed shiverer-like myelin around normal host axons, while normal oligodendrocytes produced ultrastructurally normal myelin, suggesting that the myelin defect was due to a mutation of oligodendrocytes rather than abnormal axons. This is encouraging, for it suggests that remyelination may be achieved by donor oligodendrocytes.

Based on these results, a number of studies were initiated to investigate the remyelination process in the spinal cord. There were two main lines of investigations, one which induced remyelination by implanting solid pieces of embryonic nervous tissue or cell suspensions and the other used well-defined proportions of mixed glial cell cultures.

In the former case embryonic spinal cord44,45 or brain1,46 was grafted into the spinal cord of shiverer mouse or md rat (myelin deficient rat, x-linked myelin mutant) mutants and the remyelination was detected by immunocytochemistry and electron microscopy. Normal oligodendrocytes transplanted into shiverer or mdx rat CNS can be identified immunocytochemically by the presence of myelin basic protein or proteolipid protein, respectively. Normal myelin sheaths which displayed regular periodicity were formed around axons of mutants as early as 11 days after grafting.44 Remyelinating oligodendrocytes appeared to have a tremendous capacity to migrate. They were found at remote sites from the injection and occasionally migrated up to six mm rostrocaudally.45 Acute demyelination seemed to enhance the capacity of oligodendrocytes to achieve repair: if the shiverer cord was demyelinated by lysolecithin and the transplant placed one-three segments rostrocaudally from the lesion site, remyelination by host cells occurred nine days later and donor cells migrated three-eight mm towards the lesion.46 However, the remyelination pattern was uneven. Most often only “patches” of normal myelin were observed.

At the edges of these remyelinated areas grafted oligodendrocytes often contacted abnormal host cells. In these areas there were no signs of astrocytic or microglial invasion of the grafted cells.40,44 Similarly, freshly remyelinated fibres were never seen to be attacked by macrophages even if transplanted oligodendrocytes remyelinated axons in close vicinity of macrophages.40 Nevertheless, the exact nature of these remyelinating cells was not clarified in these studies. It appeared likely that demyelination and transplantation enhanced the remyelination capability of host oligodendrocytes but host cells were not able to migrate over long distances as transplanted oligodendrocytes did. As such migration is primarily confined to glial progenitor cells47 but not mature oligodendrocytes, it was suggested that, due to the relatively low number of identifiable precursor cells in the grafted tissue, several cell types of oligodendrocyte lineage including adult progenitor cells48 could cooperate in different conditions to repair a demyelinating lesion.45,46

Factors that Determine the Success of Remyelination by Grafted Cells

A different environment for remyelinating oligodendrocytes was established by Blakemore and his coworkers. Lesions caused by application of a gliotoxic substance, ethidium bromide into the dorsal columns of the spinal cord are spontaneously repaired. However, when in addition oligodendrocyte precursors were eliminated by neonatal x-ray irradiation the lesions produced by the application of ethidium bromide were virtually free of glia and did not show spontaneous repair. Then subsequent remyelination is achieved by only the grafted glia and the extent of repair can be manipulated by the composition of the injected cells. Early experiments revealed that a mixed glial population (60-70% astrocyte, 25-40% oligodendrocyte and 5% Schwann cell) can remyelinate the demyelinated cord.49 Both Schwann cells and oligodendrocytes remyelinated the naked axons of the dorsal columns and this process spanned up to six mm rostrocaudally from the site of the graft. However, where remyelination by Schwann cells occurred (peripheral remyelination) there were no oligodendrocytes or astrocytes present while numerous astrocytes were found in areas where remyelination was achieved by oligodendrocytes (central remyelination). This suggested that astrocytes might facilitate remyelination by co-transplanted oligodendrocytes and help oligodendrocytes in their competition with Schwann cells to myelinate so that remyelination by Schwann cells can be restricted to a minimum in favour of central remyelinating processes by oligodendrocytes49 (for details on Schwann cell myelination see section: Transplantation of Schwann cells). This beneficial effect of astrocytes was confirmed in a series of studies where the proportion of oligodendrocytes and astrocytes grafted into the demyelinated cord was varied. Grafting of purified cultures of oligodendrocytes depleted of type-1 astrocytes resulted in remyelination by local Schwann cells but not by the grafted oligodendrocytes. Remyelination by grafted oligodendrocytes occured only when either the local Schwann cells were eliminated by x-ray irradiation prior to the ethidium bromide lesion or the oligodendrocyte cultures contained type-1 astrocytes.50 Thus it became evident that oligodendrocyte cultures alone are not able to remyelinate demyelinating lesions and compete with Schwann cells which invaded the lesion. Remyelination by grafted oligodendrocytes was possible only if appropriate numbers of astrocytes were transplanted along with them. But why this difference in the myelinating capacity between Schwann cells and oligodendrocytes and how do astrocytes influence remyelination?

The explanation for the different behaviour, motility and myelinating capacity of oligodendrocytes and Schwann cells came from developmental studies on glial cell precursors. Both mature Schwann cells and oligodendrocyte precursors are motile and mitotic and respond to axonal mitogens.40,47,51-53 Mature, myelin-forming oligodendrocytes are non-motile and non-mitotic,48 though they possess a considerable capacity to myelinate axons when transplanted into md rat spinal cords.54 It was suggested that the differentiation of oligodendrocytes,55 the axon-glia interaction and hence the myelination of the axon is regulated by astrocytes. Astrocytes when grafted along with oligodendrocytes are capable to limit Schwann cell invasion and remyelination probably by forming the glia limitans in the grafted cord.50,56,57 Moreover, astrocytes not only restrict Schwann cell incursion and remyelination but form an environment favourable for myelinating oligodendrocytes50 but in order to establish this normal glial environment oligodendrocyte precursors have to be present.58 However, in the absence of such co-operation, when no oligodendrocytes are co-transplanted with astrocytes, the grafted astrocyte population itself cannot prevent Schwann cell myelination.56,58 This effect is probably due to the fact that in the absence of oligodendrocytes, type-1 astrocytes, like transplanted meningeal cells clump together and form a suitable surface for migrating and myelinating Schwann cells.58-61

Electrophysiological experiments revealed that grafting of glial cells which remyelinated the cord of md rats resulted in increased conduction velocity and improved frequency-response properties of the remyelinated axons indicating a proper functional recovery.62

The number of grafted oligodendrocytes also appeared critical for the outcome of remyelination. When mixed populations of cultured astrocytes and oligodendrocytes from early postnatal (P4) brains were injected into experimentally demyelinating lesions, considerable remyelination by either Schwann cells or oligodendrocytes occurred, depending on the proportion of grafted oligodendrocytes.56 Grafts containing low proportions of oligodendrocytes (3-5%) resulted in Schwann cell remyelination while higher proportions of oligodendrocytes (10-15%) induced central (oligodendrocyte) remyelination but, again, only in the presence of co-transplanted astrocytes. Therefore, the number of oligodendrocytes and in particular their precursors appeared to play a crucial role in determining the extent of central myelination.

The Use of Oligodendrocyte Progenitors and Precursors

It is important to determine what type(s) of glial cells should be used for transplantation in order to replace missing glial cells or enhance the remyelinating capacity of the host cord. The rate of division and regenerative capacity of oligodendrocyte lineage cells decreases with differentiation, therefore earlier oligodendrocyte lineage cell types seem more appropriate candidates for successful transplantation. However, the question remains what degree of differentiation is needed to induce significant remyelination?

Very early neural progenitors isolated from the subventricular zone did not mature into myelinating oligodendrocytes following grafting into an unmyelinated lesion where they formed clusters of undifferentiated cells.63 When the progenitors were induced to become committed to the oligodendrocyte lineage in vitro, significant remyelination has been achieved following grafting. Other precursors expressing the polysialylated form of the neural cell adhesion molecule (PSA-NCAM) differentiated in vivo into oligodendrocytes and astrocytes as they did in vitro, and unexpectedly they produced Schwann cells, too.64 Oligodendrocyte progenitor cells lines produced more reliable results-they survived, migrated and remyelinated naked axons in a demyelinated spinal cord, inducing remarkable functional recovery after transplantation-but survived poorly in an intact spinal cord.65,66 It should be noted, that the grafted progenitor cells were able to reverse the functional deficit only if the axonal loss was minimal.66

Further experiments provided evidence that the better survival of grafted oligodendrocyte progenitors in the x-ray-depleted spinal cords was due to the presence of vacant “niches” created by the depleted endogenous oligodendrocyte progenitors. Oligodendrocyte “survival factors” had no effect on the survival of progenitor cells.67 Moreover, delayed availability of naked axons for the grafted oligodendrocyte progenitors also decreased their remyelinating capacity.68

The nature of the lesion also seems to influence the differentiation of glial progenitors. In situations where mainly oligodendrocytes are missing (for example md rat), transplanted bipotential O-2A cells (which are able to develop into astrocytes as well as oligodendrocytes under certain conditions) differentiate into oligodendrocytes, while in other pathological situations O-2A cells are able to produce both oligodendrocytes and astrocytes thereby reconstituting the damaged microenvironment.69

The behaviour of manipulated progenitor cell lines has also been studied when transplanted into a non-repairing spinal cord demyelinating lesion.70 If O2A bipotential progenitor cells were immortalised with the ts A58-SV40T construct and grafted into demyelinated spinal cord they established a partially remyelinating environment with the presence of astrocytes while progenitor cells continuously treated with growth factors produced a well-myelinated environment even when only few astrocytes were present. Both treatments allowed the generation of sufficient cells to achieve remyelination but none of them was similar to the normal glial environment. However, the main problem with these engineered glial population was their failure to differentiate fully and the continued capacity to divide within the graft, in particular when growth factor-treated cultures were used. It also became evident that not all members of the oligodendrocyte precursor lineage possess equally good migratory and myelinating capacities.71,72 Oligodendrocyte cultures enriched in early-phase precursors (A2B5+, O4- cells) injected into the brains of shiverer rats migrated far from the injection site and remyelinated large areas of the CNS, while transplantation of later precursor types (O4+, GC- and GC+ cells) resulted only in scattered patches of myelinated areas. Bipotential precursor cells infected with a temperature-sensitive SV40T oncogene were used to compare their in vitro and in vivo characteristics (in culture and following transplantation).73,74 The populations of precursor cells contained O4+ and GFAP+ (less then 10%) cells and at 38°C had a limited capacity to divide. This temperature-sensitive block of proliferation could be overcome by application of growth factors.74 Early passages of these cell lines yielded myelin-forming oligodendrocytes and astrocytes following transplantation into a demyelinated cord and the cells forming myelin were often present in the areas demarcated by the astrocyte-like cells. Later passages of the cell lines were not able to form myelin. These experiments showed that carefully manipulated and controlled cell lines are able to engage in complex glia-axon interaction when grafted into demyelinated cords. Nevertheless, the most difficult problem is to find predictable correlation between the in vitro and in vivo behaviour of these cell lines.71-73

The role for host oligodendrocytes in the self-repair of chemically induced demyelination (without x-irradiation) was confirmed by Blakemore and his coworkers (1991,1995).60,75 Xenogeneic (mouse) oligodendrocytes transplanted along with isogeneic rat astrocytes produced extensive remyelination in a demyelinated cord. The remyelination must have been produced by host oligodendrocytes, because, without immunosuppression, the mouse oligodendrocytes were rejected. However, the inflammation due to the rejection of the xenogeneic graft did not prevent, but augmented the repair by host oligodendrocytes, as host cells were remyelinating axons actually undergoing macrophage stripping of myelin sheaths (fig. 3).75 Similarly, experimental autoimmune inflammation reportedly promoted the survival and migration of grafted oligodendrocyte progenitors.76 Given the fact that restorative processes are usually not augmented by inflammation and accompanying immunological reactions it would be very interesting to reveal the influences which act upon adult oligodendrocytes to trigger remyelination, because to promote remyelination by host oligodendrocytes would be the logical method to repair demyelinating lesions in the CNS.

Figure 3. Following removal of immunosuppression lesions injected with mixed species cultures are infiltrated by inflammatory cells.

Figure 3

Following removal of immunosuppression lesions injected with mixed species cultures are infiltrated by inflammatory cells. When lesions from animals transplanted with mouse O2-A lineage cells and isogeneic astrocytes and maintained on cyclosporin for (more...)

Taken together, studies using experimental models of demyelination or hypomyelinating mutants have shown that mixed cultures of glial cells or newborn CNS fragments are able to achieve remyelination in areas of demyelination.77-79 Unfortunately, the exact nature of remyelinating oligodendrocytes is not clear as remyelination may be due both to differentiated oligodendrocytes and/or undifferentiated precursors. Nevertheless, an increasing weight of evidence suggests that remyelination is chiefly due to migrating oligodendrocyte precursors though the role of adult oligodendrocytes cannot be neglected.54,78 Possible therapies to achieve complete remyelination in demyelinating lesions are of extreme importance. At present it is not certain whether or not the best cure to remyelinate such lesions will be oligodendrocyte transplantation. An alternative solution and way of future research would be to encourage remyelination by adult host oligodendrocytes and this opportunity appears almost as promising as grafting cultured oligodendrocytes. Nevertheless, the glial environment and the interaction between astrocytes and oligodendrocytes seem crucial for remyelinating oligodendrocytes59,78 therefore this aspect of human neuropathology needs to be further studied.

Remyelination Induced by Grafted Stem Cells

The limited availability of myelinating cells that can be used for transplantation urged researchers to study the myelinating capacity of stem cells. Stem cells used in these studies derived either from early embryos (embryonic stem cells)80 or from later stages81 including adult human brain-derived stem cells.82 The stem cells were clonally expanded, embryonic stem cells needed induction to differentiate by retinoic acid. Although the cells maintained a versatile phenotype prior to grafting in culture, interestingly all the investigated stem cells turned into myelinating cell types in a dysmyelinated or amyelinated mutant rodent spinal cord after grafting. They produced either a CNS-type or a PNS-type myelin sheath indicating, that some of the neural precursor cells retain the capability to differentiate into cells with morphological characteristics of Schwann cells.82 In one case the remyelinated axons were tested electrophysiologically and showed near normal conduction velocities. Briefly, it can be concluded that stem cells or clonal neural precursor cells are able to produce remyelination in the demyelinated spinal cord, although several questions remain to be addressed. Most importantly the controlled in vitro differentiation of the well-characterised neural precursor cell types should be precisely determined in order to avoid any unexpected outcome of the prospective transplantation trials in humans.

Transplantation of Astrocytes

Considerations that Led to Grafting Astrocytes into the Spinal Cord

Although it was suggested by Ramon y Cajal, that the failure of regeneration in the CNS of mammals was probably due to the glial scar formed after injury, only studies in the past few years have clarified some features of the effects of glia on axonal growth. Our knowledge about the regenerative capacity of the spinal cord of different species has been quite well-established in the 1960s, well before we knew much about the behaviour of reactive glial elements which play a role in the regulation of regenerative processes. According to classical views the regeneration of the adult mammalian spinal cord was non-existent, since after lesioning of the spinal cord neither function nor structure was restored. In contrast, in lower vertebrates, such as in lamprey, bony fish and goldfish return of the motor function of the transected spinal cord was observed.83 Although functional recovery did occur, morphological restoration was incomplete. In the lamprey and young goldfish the regenerative axon growth observed was not prevented by the neuroglial scar83 while in adult mammals the glial scar formed a barrier for regenerating axons.84 The phylogenetic dichotomy on spinal cord regeneration was further complicated when significant regeneration was found in transected spinal cord of neonatal rats.85 Also little gliosis was found in the injured spinal cord of neonatal rats (for more details see Chapter 3).85

These findings led to the conclusion that in the case of successful regeneration in primitive chordates and young goldfish the return of motor function (for example swimming) is only partially due to the reorganization of spinal cord circuitry and probably glial elements also contribute to the improvement of function either by facilitating or simply not preventing axonal growth or perhaps exerting a trophic action on spinal cord neurones. It has also been shown that mature astroglia inhibit whilst immature astrocytes facilitate axonal growth.86-88 The reactive astrocytes are also not unanimously inhibitory in nature: reactive astrocytes secondary to a penetrating trauma (anisomorphic astrocytes) are more permissive for axonal growth than astrocytes responding to Wallerian degeneration (isomorphic astrocytes).89 In contrast to the fact that reactive astrocytes are thought to inhibit axonal growth, they seem to facilitate neuronal survival in vitro through the release of soluble factors.90 This effect could be of particular importance when reactive microglia which are known of their neurotoxic activity penetrate the areas of CNS injury.90

The putative trophic function of glial cells was also suggested when embryonic CNS tissue grafted into the brain has been shown to reverse deficits in spatial learning and memory91 and improve behavioral assymetries caused by chemically induced catecholamine depletion.92,93 Memory deficits could be improved just as effectively by grafted astrocytes from in vitro cultures as by fetal cortical transplants.94 The interest in revealing the properties of grafted astrocytes resulted in a series of experiments where astrocytes were grafted alone or co-grafted with other glial cells into the spinal cord. The mammalian spinal cord proved a very good model for studying regenerative events as its elongated structure with several long fibre tracts allows the lesion as well as the subsequent intervention to be applied at various levels of the spinal cord.

Migration and Effects of Grafted Astrocytes

After successful attempts of grafting astrocytes into the brain, Bernstein and colleagues began to study the behaviour and migration pattern of the grafted and prelabelled astrocytes. In their experimental model E14 embryonic neocortex95-99 or E14 spinal cord100 was grafted into the thoracic spinal cord. The astroglial cells, prelabelled with Phaseolus vulgaris leucoagglutinin were identified by double-labelling for their GFAP content and were found to migrate as far as the lumbar spinal cord or dorsal column nuclei in the medulla.95-96 The furthest distance from the graft where double-labelled astrocytes were found 90 days after transplantation was 55 mm suggesting a 0.72-0.76 mm/day migration rate. The primary direction of migration was rostrocaudal and migrating astrocytes mainly used the long fibre tracts, in particular the dorsal column tracts and glia limitans as pathways. Only limited migration was observed laterally in the spinal grey matter (up to 1 mm). Two functions were ascribed to the migrating astrocytes. Since grafted astrocytes appeared along the borders of the graft as early as seven days after transplantation when the grafted tissue itself contained no astrocytes, they were thought to have provided a migratory pathway for neurones of graft origin to penetrate the host tissue.98 Indeed, when E14 embryonic cortex was transplanted into the dorsal horn of the spinal cord, VIP (Vasoactive Intestinal Peptide) immunoreactive neurones, which are normally absent from the spinal cord, were observed close to motoneurones.97 Interestingly, the reactive astrogliosis began one month after transplantation in the centre of the graft, suggesting that in this case the onset of gliosis corresponds to the termination of dendritic development. Such a delay in the onset of astrocytosis was thought to be sufficient for differentiation and migration of neurones.97 The other function believed to be fulfilled by migrating astrocytes is merely trophic. Lesion to the fasciculus gracilis at cervical level results in loss of proprioception and deficits in hindlimb placement as well as atrophy of neurones in the dorsal column nuclei.101,102 This deficit could be ameliorated by placing a solid piece of embryonic cervical spinal cord into the lesion cavity.99,101,102 Nevertheless, no morphological reinnervation of the nucleus gracilis by grafted neurones occurred, i.e., the fasciculus gracilis was not repopulated by axons from the graft. Since astrocytes of graft origin were found in the nucleus, their possible trophic influence in maintaining the host neurones in the nucleus gracilis was suggested.101,102 The nature of this trophic effect, however, remains to be determined. Attempts to do so revealed high levels of nerve growth factor (NGF) in the nucleus gracilis of injured animals 90 days after injury, but only slightly elevated NGF levels were found in animals in which the lesion was corrected with embryonic grafts.103 Therefore NGF seems to be detrimental to neuronal maintenance and return of hindlimb functions, since increase of NGF was prevented possibly by migrated astrocytes and high NGF levels did not result in neuronal maintenance in the lesioned nucleus gracilis. The active presence of other factors exerted by grafted astrocytes is still far from proven.

Different results were obtained when cultured and purified astrocytes were grafted into the lesion cavity. Although they migrated to the nucleus gracilis, unlike astrocytes originating from solid tissue grafts they failed to prevent atrophy of the cluster neurones and improve hindlimb functions.102 This finding was surprising because similar grafts of purified astrocytes placed into the cortex attenuated behavioral dysfunctions after frontal cortex lesion in rats.94 Moreover, Wrathall et al94 in 1985 reported that purified immature astrocytes grafted into the contused spinal cord have improved the functional deficits in rats. Wang et al (1995) grafted cultured astrocytes into the hemisected adult spinal cord.105 Grafted astrocytes not only induced the decrease of the volume of scar tissue in the hemisected cord, but promoted the axonal growth around and into the scar tissue,too.105 The grafted astrocytes migrated from the lesion site at a rate of 0.6 mm/day. It was not clear, whether these beneficial effects of grafted astrocytes resulted in improved functional recovery.

It cannot yet be determined which astrocytic factors may play role in maintaining or rescuing lesioned neurones: embryonic (immature) astrocytes may express cell surface molecules or produce trophic substances and both of these may be lost or modified during culturing or purification.102 However, one should be aware that different purification and culture methods may result in loss of different surface molecules or may differentially alter the behaviour and maturation of astrocytes.

Another approach to transplantation of astrocytes into the spinal cord was used by Blakemore and his group.57,59,106 These authors were interested in the repair of demyelinated lesions. The capacity of immature astrocytes to alter the microenvironment of an artificial demyelinating lesion and regulate the remyelination pattern achieved by Schwann cells and oligodendrocytes seems to provide a useful tool to achieve repair. Several in vitro and in vivo studies in the PNS107 have demonstrated that astrocytes may form an environment suitable for growing axons and remyelinating oligodendrocytes. Accordingly, if type-1 astrocytes are transplanted into a lesioned spinal cord where the demyelinating lesion was produced by ethidium bromide and x-ray irradiation, the grafted astrocytes integrated with the damaged host tissue.57 Moreover, a subsequent remyelination in the lesioned cord occurred which was due to the host oligodendrocytes. These cells normally have limited myelinating capacity, but the environment produced by grafted astrocytes may have induced them to increase their ability to produce myelin. Astrocytes may secret mitogenic (platelet derived growth factor) and possibly migratogenic factors which may stimulate adult host progenitor cells to remyelinate. The nature of this influence of astrocytes on the environment i.e., the extracellular matrix was studied by Franklin et al106 In their experiments completely demyelinated lesions in the spinal cord were replenished by cultured and purified astrocytes. The grafted cell suspensions were carefully purified in order to remove oligodendrocytes and Schwann cells. The transplanted astrocytes were able to establish an astrocytic environment which contained an integrated astrocyte matrix, i.e., demyelinated axons surrounded with robust astrocytic processes. This indicated that the grafted immature astrocytes migrated, differentiated into a mature-like astrocyte type and integrated with the host tissue. Moreover, this environment formed by grafted astrocytes was very similar to the chronic demyelinated plaques of multiple sclerosis. In other studies the environment-forming effects of astrocytes derived from different sources were studied.108 “Type-1” astrocytes derived from rat tissue cultures formed cords while “type-2”astrocytes, which differentiated from O-2A progenitor cells spread through a glia-free environment and thus showed greater capacity to fill glia-free lesions than tissue culture astrocytes. Similarly, progenitor-derived astrocytes filled infarcted areas of the spinal cord white matter more effectively than tissue culture astrocytes, although none of them was able to induce axonal growth into the reconstituted lesion site.

It appears from these results that astrocytes are responsible for the maintenance of the appropriate environment and physical framework within the CNS and facilitate the interaction between neurones and oligodendrocytes. However, the regulatory function of grafted astrocytes seems to be more complicated. Recent results indicate that astrocytes have a very strong stimulating influence on oligodendrocyte differentiation during development in vitro and are able to overcome the potent inhibitory effects of basic fibroblast growth factor.55 Astrocytes also have a strong influence on the remyelinating capacity of co-transplanted oligodendrocytes and Schwann cells (for details see section: Transplantation of oligodendrocytes).

Transplantation of Macrophages into the Injured Spinal Cord

The CNS has been regarded as an “immunologically privileged” site, where there is little immunological survey of the brain and spinal cord. However, recently this view has been significantly modified, suggesting that the immune system, under specific circumstances, such as in case of infections or tumors is indeed involved into the response to these processes. The most important components of this limited immunological response are brain macrophages. Macrophages are known to remove debris after injury, to secrete cytokines and thus regulating mitogenic and chemotactic activities within the affected tissues (for review see Lotan and Schwartz 1994,109 Schwartz et al 1999110). These potential “renewing” activities of the macrophages together with the limited inflammatory response of the CNS following injury suggested that grafting of macrophages into a lesioned spinal cord may be beneficial. Indeed, macrophages exposed ex vivo to regenerating peripheral nerves and then grafted into the lesion site of complete spinal cord transection induced some recovery in paraplegic rats and the functional recovery was accompanied by improved electrophysiological activity.111 Morphologically descending fibres were seen passing the site of transection. Repeated transection of the fused lesion site led to the loss of recovery, indicating that improved function was due to the re-established connections between proximal and distal spinal cord stumps.

Other studies have also shown the neurite-promoting activity of microglial grafts.112-114 The above studies performed detailed immunohistochemical and molecular biological analysis and suggested that microglial cells may promote axonal regeneration

  1. by rapidly degrading myelin proteins and
    by promoting the synthesis of growth-promoting extracellular molecules.

This latter effect may be due to residing glial cells and Schwann cells that migrated into the lesion. Treatment of the grafted macrophages with Macrophage Inhibitory Factor prior to grafting resulted in reduced axonal growth-promoting activity of microglial grafts (fig. 4).112

Figure 4. In these transverse sections the presence of CGRP-immunoreactive axons (arrows in A, C, and D) and ED-1 immunoreactive phagocytic cells (arrowheads in B and E) can be found in the transplant tissue (T) adjacent to the nitrocellulose implant (N).

Figure 4

In these transverse sections the presence of CGRP-immunoreactive axons (arrows in A, C, and D) and ED-1 immunoreactive phagocytic cells (arrowheads in B and E) can be found in the transplant tissue (T) adjacent to the nitrocellulose implant (N). Adjacent (more...)

Although further thorough studies are needed to reveal the role of macrophages in spinal cord regeneration, the recent studies on macrophage/microglia transplantation suggest that grafted microglial cells are able to alter the microenvironment of the injured spinal cord and thus may promote axonal growth.

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