Schwann Cell Transplantation to Remyelinate the Spinal Cord

Transplantation of SCs into the demyelinated rodent spinal cord results in remyelination with a characteristic peripheral pattern (Blakemore and Crang, 1985; Baron-von Evercooren et al., 1992; Honmou et al., 1996). Moreover, when anatomical repair is achieved subsequent to SC transplantation, near-normal conduction velocity of the remyeli-nated axons is achieved (Honmou et al., 1996). Endogenous remyelination of demyelinated CNS axons by oligodendro-cytes (Gledhill et al., 1973) or SCs (Blight and Young, 1989; Felts and Smith 1992) results in the reestablishment of relatively normal impulse conduction in animal models of demyelination. However, endogenous remyelination is very limited in human demyelinating diseases such as MS (Prineas and Connell, 1979). Given the success of cell transplantation to form functional myelin in animal models, myelin-forming cell transplantation has been suggested as a potential repair strategy for demyelinated CNS axons.

Transplantation of human SCs derived from human sural nerve can remyelinate spinal cord axons in the immunosup-pressed rat (Kohama et al., 2001). In these experiments a focal demyelinated lesion was created in the dorsal column of the spinal cord of 12-week-old rats by X-irradiation and ethidium bromide injection (X-EB) (see Kohama et al., 2001, for technical details). This lesion presents as a persistent area of demyelination that lacks astrocytes. An example of a remyelinated axon region of the spinal cord 3 weeks after focal injection of reconstituted cryopreserved human SCs is shown in Fig. 1. Note the relatively large number of myelinated axons with typical SC morphology (i.e., large cytoplasmic and nuclear regions). Electron micrographs (not shown) reveal the presence of a basement membrane and extracellular collagen deposition. The conduction velocity of the axons remyelinated by the human SCs was improved (Fig. 2), indicating that electrophysiological function of the remyelinated axons was improved.

Autologous tissue represents one possible source of SCs for transplantation into patients with demyelinating disease.

Figure 1 Remyelinated axons after human Schwann cell transplantation showing a peripheral pattern of myelination. Photomicrograhs were obtained from spinal cords placed in fixative after in vitro electrophysiological recordings. (A) Lesion area of dorsal columns 3 weeks after induction of the X-EB lesion. Sg refers to substantia gelatinosa of in the dorsal horn. (B) Higher power micrograph from the boxed region of the lesion showing remyelinated axons. Arrows in B indicate examples of axons myelinated by cells with large nuclear and cytoplasmic domains characteristic of peripheral myelin. Calibration in B corresponds to 100 |m in A and 10 |m in B. (Modified from Kohama et al., 2002.)

Figure 1 Remyelinated axons after human Schwann cell transplantation showing a peripheral pattern of myelination. Photomicrograhs were obtained from spinal cords placed in fixative after in vitro electrophysiological recordings. (A) Lesion area of dorsal columns 3 weeks after induction of the X-EB lesion. Sg refers to substantia gelatinosa of in the dorsal horn. (B) Higher power micrograph from the boxed region of the lesion showing remyelinated axons. Arrows in B indicate examples of axons myelinated by cells with large nuclear and cytoplasmic domains characteristic of peripheral myelin. Calibration in B corresponds to 100 |m in A and 10 |m in B. (Modified from Kohama et al., 2002.)

Presumably, SCs are not antigenically predisposed to the immunological attack seen in MS as are oligodendrocytes. The demonstration of anatomical and electrophysiological repair of demyelinated axons by adult human SCs is an important prerequisite for future consideration of these cells as candidates for autologous transplantation studies in humans. One potential problem with the use of SCs to remyelinate lesions in patients with MS is the presence of a glial scar in MS lesion sites that could limit cell migration and remyelination potential. In the X-EB lesion in the rat where relatively extensive remyelination is observed, it is

Figure 2 Intra-axonal recordings from demyelinated and remyelinated dorsal column axons. (A) Schematic showing arrangement of intra-axonal recording and stimulation sites. Intra-axonal recordings were obtained from dorsal column axons outside of the lesion where the axons were normally myelinated. Stimulating electrodes were positioned outside (S1-S2) and within (S3-S4) the X-EB lesion zone to assess single axon conduction velocity over both the demyelinated or remyelinated axon segment and the normally myelinated axon segment of the same axon. (B) Pairs of action potentials recorded from S1-S2 stimulation (1), S3-S4 in the demyelinated dorsal columns (2), and S3-S4 following cell transplantation (3). Recordings were obtained at comparable conduction distances. (C) Plot of the conduction velocity of axon segments within the lesion (S3-S4) versus conduction velocity of the axon segment outside of the lesion (S1-S2) for X-EB lesioned spinal cord without (open circles) and with (closed squares) transplantation. (Modified from Kohama et al., 2002.)

Figure 2 Intra-axonal recordings from demyelinated and remyelinated dorsal column axons. (A) Schematic showing arrangement of intra-axonal recording and stimulation sites. Intra-axonal recordings were obtained from dorsal column axons outside of the lesion where the axons were normally myelinated. Stimulating electrodes were positioned outside (S1-S2) and within (S3-S4) the X-EB lesion zone to assess single axon conduction velocity over both the demyelinated or remyelinated axon segment and the normally myelinated axon segment of the same axon. (B) Pairs of action potentials recorded from S1-S2 stimulation (1), S3-S4 in the demyelinated dorsal columns (2), and S3-S4 following cell transplantation (3). Recordings were obtained at comparable conduction distances. (C) Plot of the conduction velocity of axon segments within the lesion (S3-S4) versus conduction velocity of the axon segment outside of the lesion (S1-S2) for X-EB lesioned spinal cord without (open circles) and with (closed squares) transplantation. (Modified from Kohama et al., 2002.)

important to note that the lesion is agliotic, and thus the potential impediment of gliosis is not an issue. However, SCs transplanted into a contusion injury model in the spinal cord where gliosis does occur lead to increased myelination, axon sparing/regeneration, and improved functional outcome in rodents (Takami et al., 2002).

Experimental in vitro studies have shown that astrocytes inhibit both SC proliferation and SC myelination potential (Guenard et al., 1994). When SCs are transplanted into the demyelinated spinal cord (Blakemore et al., 1986), they remyelinate axons throughout the area from which astrocytes are absent but not beyond. On the basis of this type of observation, it has been suggested that SCs and astrocytes are mutually exclusive. In contrast, OECs coexist with astrocytes within the olfactory bulb, are associated with olfactory receptor neurons from their peripheral origin to their central projection in the outer nerve layer of the olfactory bulb, and share in the formation of the glia limitans (Doucette, 1984, 1991). One assumption favoring the use of OECs in CNS repair studies, therefore, was that the use of OECs instead of SCs might prevent the astrocytic response to nerve injury (Franklin and Barnett, 1997, 2000), which is thought to block the regeneration response. Studies including the co-culture of either SCs or OECs with astrocytes have shown that OECs, in contrast to SCs, intermingle with astrocytes (Lakatos et al., 2000). Moreover, contact with SCs, but not with OECs, induced apoptosis of astrocytes (Lakatos et al., 2000). After induction of an electrolytic lesion of the rat corticospinal tract, injection of OECs induced axonal elongation across and beyond the lesion, and a reduced upregulation of GFAP expression in the adjacent astrocytic processes compared with nontransplanted lesions (Li et al., 1997; 1998). The assumption emerging from these studies that OECs in contrast to SCs might reduce astrocytosis in vivo was recently tested by application of OECs into the photochemically lesioned spinal cord (Verdu et al., 2001). It was found that OECs reduced the number of hypertrophic astrocytes as well as the maximal area and volume of the cystic cavity (Verdu et al., 2001) compared to nontransplanted controls.

III. Transplantation of Olfactory Ensheathing Cells (OECs) to Remyelinate the Spinal Cord

Adult olfactory receptor neurons continually undergo turnover from an endogenous progenitor pool, and their nascent axons grow through the olfactory nerves and cross the PNS-CNS interface where they form new synaptic connections in the olfactory bulb (Graziadei et al., 1978). This apparent support role of OECs in axonal growth in the adult CNS has spawned extensive research aimed at studying the potential of OEC transplants to encourage axonal regeneration and functional recovery in spinal cord injury models (Li et al., 1997, 1998; Ramon-Cueto et al., 1998, 2000; Imaizumi et al., 2000a, 2000b). In the normal nervous system, OECs surround or ensheath bundles of nonmyelinated axons in the olfactory nerve and do not form myelin. OECs share with SCs the ability to support axonal ensheathment and regrowth, but they do not share the same developmental origin or some immunocy-tochemical and morphological features of SCs (Ramon-Cueto and Valverde, 1995). The SC-like morphology of OECs includes p75 and S100 expression, as well as a spindle-shaped morphology in culture (Pixley, 1992).

A large body of work supports the proposal that transplantation of OECs into various spinal cord injury and demyelination models can promote axonal regeneration, remyelination, and functional recovery (Franklin et al., 1996; Li et al., 1997, 1998; Ramon-Cueto et al., 1998, 2000; Imaizumi et al., 2000a, 2000b; Lu et al., 2002; Keyvan-Fouladi et al., 2003; Plant et al., 2003). Yet, there is an important controversy as to whether the transplanted OECs associate with axons and form peripheral myelin, as opposed to recruiting endogenous SCs that form myelin (Takami et al., 2002; Boyd et al., 2004). OECs can express a number of trophic factors, transcription factors, and extracellular matrix molecules (Ramon-Cueto and Avila, 1998; Chuah and West, 2002; Au and Roskams, 2003; Ramer et al., 2004), which could facilitate endogenous SC cell invasion, angio-genesis, and activation of progenitor cells to facilitate repair.

A recent study failed to observe myelination in vitro in a co-culture experiment with dorsal root ganglion neurons and immunoselected (p75) OECs under culture conditions permissive for myelination by SCs (Plant et al., 2002). These investigators raise the important question as to whether transplanted OECs might induce or enhance the migration of endogenous SCs into the transplantation site (Brook et al., 1998). Moreover, although numerous reports suggest that OECs can form myelin when transplanted into the demyelinated (Franklin et al., 1996; Imaizumi et al., 1998; Barnett et al., 2000; Kato et al., 2000; Akiyama et al., 2004; Radtke et al., 2004) or injured spinal cord (Li et al., 1997, 1998; Imaizumi et al., 2000a, 2000b), one study was unable to find evidence of OEC myelination in the compressed spinal cord, suggesting that OEC (derived from embryos) transplantation may facilitate endogenous SC invasion into the lesion site (Boyd et al., 2004). To address this issue, we prepared cell suspensions of OECs from the olfactory bulb of alkaline phosphatase expressing adult transgenic rats (Kisseberth et al., 1999). The marker gene, human placental alkaline phosphatase

(hPAP), is linked to the ubiquitous active R26 gene promoter, and its stable expression has been demonstrated by neural precursor cells in culture and after transplantation into the CNS (Mujtaba et al., 2002; Han et al., 2002). Transplantation of cell suspensions enriched in adult OECs (>95% p75+ and S100+) derived from hPAP transgenic rats can be readily identified in vivo and are associated with myelin formation (Fig. 3) (Akiyama et al., 2004). The extensive degree of remyelination by identified SCs and OECs indicates that both cell types under appropriate in vivo conditions are capable of forming myelin in the spinal cord.

In rodents, considerable endogenous remyelination can occur after development of a chemically-induced demyeli-nating lesion (Akiyama et al., 2004). A large number of myelinated profiles are present by 3 weeks after induction of focal demyelinating lesions in the rat spinal cord (Akiyama et al., 2004). Figure 4 shows a progressive increase in conduction velocity from 1 to 6 weeks after a focal injection of ethidium bromide (EB) into the rat spinal cord. When the spinal cord is X-irradiated 3 days before EB injection to block mitosis of progenitor cells and endogenous repair, a persistent region of demyelination is observed even at 6 weeks postinjury. The conduction velocity of the persistently demyelinated axons remains low (about 1.0 m/sec) throughout this period. However, when SCs or OECs are transplanted into the X-EB lesion, one can see that, for both cell types, conduction velocity is improved at both 3 and 6 weeks posttransplantation. In this lesion model system, improvement in conduction velocity was comparable after transplantation of SCs and OECs.

Most of the experimental work showing axonal repair using OECs was done on the rodent system, and the OEC preparations were of varying purity and cellular composition. The robust capability of rodents in terms of endogenous myelin repair may differ in primates. Unlike what occurs in the rodent, very little endogenous repair was observed after EB lesions in the nonhuman primate spinal cord at 4 weeks postinjection (Radtke et al., 2004). However, after grafting of OECs derived from a transgenic pig model expressing H-transferase to alter carbohydrate structure of the cells to mimic that of the human Type O blood group, considerable peripheral-like myelin was observed in the primate spinal cord (Fig. 5). These results suggest that although endogenous repair of myelin may be less robust in primates than in rodents, transplantation of peripheral-myelin forming cells are capable of remyelinat-ing primate spinal cord axons.

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