12 M. Dezawa Anatomical Science International (2002) 77, 12–25

Review Article

Central and peripheral nerve regeneration by transplantation of Schwann cells and transdifferentiated bone marrow stromal cells Mari Dezawa Department of Anatomy, Yokohama City University School of Medicine, Yokohama, Japan

Abstract In contrast to the peripheral nervous system (PNS), little structural and functional regeneration of the central nervous system (CNS) occurs spontaneously following injury in adult mammals. The inability of the CNS to regenerate is mainly attributed to its own inhibitorial environment such as glial scar formation and the myelin sheath of oligodendrocytes. Therefore, one of the strategies to promote axonal regeneration of the CNS is to experimentally modify the environment to be similar to that of the PNS. Schwann cells are the myelinating glial cells in the PNS, and are known to play a key role in Wallerian degeneration and subsequent regeneration. Central nervous system regeneration can be elicited by Schwann cell transplantation, which provides a suitable environment for regeneration. The underlying cellular mechanism of regeneration is based upon the cooperative interactions between axons and Schwann cells involving the production of neurotrophic factors and other related molecules. Furthermore, tight and gap junctional contact between the axon and Schwann cell also mediates the molecular interaction and linking. In this review, the role of the Schwann cell during the regeneration of the sciatic (representing the PNS) and optic (representing the CNS) nerves is explained. In addition, the possibility of optic nerve reconstruction by an artificial graft of Schwann cells is also described. Finally, the application of cells not of neuronal lineage, such as bone marrow stromal cells (MSCs), in nerve regeneration is proposed. Marrow stromal cells are known as multipotential stem cells that, under specific conditions, differentiate into several kinds of cells. The strategy to transdifferentiate MSCs into the cells with a Schwann cell phenotype and the induction of sciatic and optic nerve regeneration are described. Key words: cell transplantation, gap junction, marrow stromal cells, stem cells, tight junction.

Introduction The ability of the mammalian peripheral nervous system (PNS) to regenerate axons after injury is well documented (Zochodne, 2000). Results of studies in the past decade suggested that the Schwann cell is one of the most important components of the peripheral glia that forms myelin and plays a key role during the regeneration. The process of Wallerian degeneration and subsequent regeneration is accompanied by the proliferation and activation of Schwann cells, which then produce various kinds of factors and other related molecules so that the axons of the proximal nerve stump grow through the distal stump in close contact with these Schwann cells (Martini, 1994; Hall, 2001). On the other hand, the successful Correspondence: Mari Dezawa, Department of Anatomy, Yokohama City University School of Medicine, 3–9 Fukuura, Kanazawa-ku, Yokohama 236–0004, Japan. Email: [email protected] Received 7 January 2002; accepted 7 January 2002.

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elongative axonal regeneration and functional reconstruction do not normally occur in the central nervous system (CNS) of adult mammals (Aguayo, 1985). A number of factors are thought to contribute to this lack of recovery, including inhibitorial glial scar formation by astrocytes, myelin inhibition by oligodendrocytes, cell death, insufficient support of growth factors and the lack of permissive substrates for axonal growth (Dezawa et al., 1999; Schwartz et al., 1999; Lacroix & Tuszynski, 2000; Asher et al., 2001; Fournier and Strittmatter, 2001). However, remarkable progress in molecular biology and neuroscience have revealed that CNS neurons originally have the intrinsic ability to regenerate, which can be triggered by proper artificial treatments under adequate circumstances (Iwashita et al., 1994; Quan et al., 1999; Fouad et al., 2001; Jones et al., 2001). A strategy for eliciting CNS regeneration is to adopt some of the favorable properties exhibited in the PNS, which can be achieved by providing neurotrophic factors and the transplantation of cells known to support axonal regeneration. Above all, the Schwann cell is a powerful candidate to be

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Nerve regrowth by cell transplantation transplanted because axons are known to regenerate when the glial milieu is experimentally replaced by Schwann cells and/or peripheral nerve segments (So and Aguayo, 1985; Berry et al., 1988; Vidal-Sanz et al., 1991; Raisman, 1997). Indeed, several experiments in the spinal cord and some other areas within the CNS have shown that either the injection or transplantation of a polymer tube filled with cultured Schwann cells improved axonal growth across the injured site (Bunge, 1994; Harvey & Plant, 1995; Harvey et al., 1995; Negishi et al., 2001; Oudega et al., 2001). Moreover, the Schwann cell is able to reconstruct myelination after the induction of axonal regeneration (Martini, 2001). Thus, Schwann cells provide an environment suitable not only for the regeneration of the PNS itself but also for the reconstruction of CNS. This review firstly focuses on the interaction between the regenerating axon and the Schwann cell both in sciatic nerve regeneration (representing the PNS), and in optic nerve regeneration (representing the CNS) induced by the transplantation of a PNS segment in the adult rat. The optic nerve is a readily accessible CNS tract that mostly contains glial cells and axons arising from retinal ganglion cells (RGCs), and that cell bodies and axons of RGCs can be manipulated separately. Such relative anatomical simplicity has made it a valuable subject for the investigation of axon–glia interactions during regeneration. Some examples and evidence are described about the direct and dynamic communication that exists between regenerating axon and the Schwann cell, which might play an important role in the mechanism of PNS and CNS nerve regeneration. In addition, the possibility of whether an artificial graft composed mainly of Schwann cells provides a favorable environment for the reconstruction of optic nerve circuit is discussed. An artificial graft induced the regrowth of transected optic nerve fibers over several centimeters into superior colliculus, the terminal projection site of optic nerve fibers in the CNS. The histological and functional analysis of those regenerated optic nerve fibers are described. Further, the application of cells not of neuronal lineage on nerve regeneration is proposed. Bone marrow stromal cells (MSCs) are well known as multipotential stem cells that, under specific conditions, differentiate into several types of cells such as osteoblasts, adipocytes, chondrocytes and muscles (Prockop, 1997). Although MSCs are quite distinct from neuronal lineage, the potential of MSCs to develop both in vivo (Eglitis & Mezey, 1997; Kopen et al., 1999) and in vitro (Sanchez-Ramos et al., 2000; Woodbury et al., 2000, Deng et al., 2001) into neurons and astrocytes has been reported. In this review, the evidence from recent work on the possibility of MSCs to transdifferentiate into the cells with

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a Schwann cell phenotype and the application of those cells to the neural regeneration are discussed.

Regeneration of the PNS Injury to the PNS produces a well-characterized cascade of cellular and molecular events in the nerve stump distal to the site of injury. These processes are collectively known as Wallerian degeneration (Fawcett & Keynes, 1990; Torigoe et al., 1996; Hall, 2001). In the earlier processes, axons degenerate and the myelin sheaths break up into ovoids, but they are phagocytosed by Schwann cells and macrophages invade the degenerating nerves. The characteristic feature of Wallerian degeneration is the proliferation and activation of Schwann cells remaining within the distal nerve stump, which results in the formation of Schwann cell cordons. Proximal to the site of injury, damage to nerve axons immediately influences the intracellular signaling pathway, which subsequently induces the expression of various kinds of genes related to the cell emergency, including c-fos and c-jun (Soares et al., 2001). On the other hand, axons give rise to one or more growth cones. The growth cones enter into the Schwann cell tubes, which form the pathway for regenerating axons to the targets. Early in the process, one Schwann cell surrounds several regenerating axons, but eventually they segregate to form a 1:1 relationship between the axon and Schwann cell, and the process of remyelination is completed (Fawcett & Keynes, 1990; Hall 2001). Schwann cells have a reciprocal relationship with the axons they ensheathe. Axonal signals, whether acting by direct contact or by diffusible factors, regulate the gene of Schwann cells and control proliferation and differentiation. Schwann cell signals regulate, and vice versa, gene expression and intracellular signaling of axons (Bolin & Shooter, 1993). Such a tightly regulated relationship of axons and Schwann cells is disrupted by injury, and in the subsequent Wallerian degeneration, Schwann cells quickly downregulate the expression of myelin protein genes and upregulate the low affinity neurotrophin receptor p75, growth associated protein 43 (GAP-43), and then start to dedifferentiate and divide (Hall, 2001). Neurotrophic factors are key regulatory proteins that modulate neuronal survival, axonal growth, synaptic plasticity and neurotransmission. Schwann cells produce various kinds of trophic factors and receptors including neurotrophins, ciliary neurotrophic factor (CNTF), transforming growth factor, basic fibroblast growth factor (bFGF) and glial cell line-derived neurotrophic factor, which contribute to successful axonal regeneration

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(von Bartheld, 1998; Abe et al., 2001; Hall, 2001). Denervated Schwann cells also express a variety of cell adhesion molecules including neural cell adhesion molecules (NCAM), L1 and N-cadherin (Martini, 1994) and integrins represented by b1-integrin that mediate interaction between the Schwann cell and axons, including growth cones (Chernousov & Carey, 2000). Besides these trophic factors and cell adhesion molecules, the Schwann cell produces molecules of the extracellular matrix such as fibronectin, laminin J1/tenascin and merosin (laminin-2) to injured axons that attach and extend their processes (Chernousov & Carey, 2000). Reinnervated Schwann cells cease dividing, downregulate expression of the above molecules and revert to an axon-associated phenotype (Hall, 2001). Collectively, nerve regeneration might be induced by the integrated functions of these factors and the activated Schwann cells.

Optic nerve regeneration elicited by peripheral nerve transplantation It has been recognized that regeneration of the CNS in adult mammals is quite restricted. Once the CNS is damaged, they are exposed to very severe circumstances for regeneration. A number of factors are involved in the lack of regeneration including myelin inhibition by oligodendrocytes, glial scarring, neuronal cell apoptosis and insufficient trophic factor support (Dezawa et al., 1999; Schwartz et al., 1999; Lacroix and Tuszynski, 2000; Asher et al., 2001; Fournier and Strittmatter, 2001). Oligodendrocytes, which represent the myelinating glia in the CNS, carry axon growth-inhibiting molecules on their surface (Brittis & Flanagan, 2001; Fouad et al., 2001; Fournier & Strittmatter, 2001). Supporting this idea, the loss of regeneration capacity during development correlates roughly with the onset of myelination (Fournier & Strittmatter, 2001). Moreover, the phenomenon described as ‘contactmediated inhibition’ came from the in vitro observation that when growth cones encountered oligodendrocytes, firm filopodial contact was sufficient to induce a rapid and long-lasting arrest of the growth cone motility, often followed by a collapse of the structure (Schwab & Caroni, 1988). Even when optimal levels of neurotrophic factors are provided, axonal growth is not possible if inhibitory myelin components surround the axons (Cadelli et al., 1992). Some components of the myelin produced by oligodendrocytes, Nogo and myelin associated glycoprotein (MAG) are identified to inhibit axonal growth, and antibodies against these proteins resulted in axonal regrowth and functional recovery in the

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CNS (Shen et al., 1998; Cai et al., 1999; Fournier & Strittmatter, 2001). Astrocytes form a glial scar and actively block axonal growth (Nieto-Sampedro, 1999; Asher et al., 2001). Such reactive gliosis is characterized by the hypertrophy and proliferation of astrocytes. Axons do not extend through the scar and axonal tips form club-like structures that can remain in place for years. The expression of repulsive molecules such as semaphorin-3A, tenascin and chondroitinsulfate proteoglycans are related to their repulsive nature (Mckeon et al., 1991; McMillian et al., 1994; Pasterkamp et al., 1999). Recent research has suggested that reactive glial extracellular matrix is more directly associated with the failure of axonal regeneration, and that myelinated white matter beyond the glial scar is rather permissive for regeneration (Davies et al., 1997). Peripheral macrophages are related to the process of PNS degeneration and subsequent regeneration, and macrophages and/or their associated substances, such as cytokines and secreted factors, stimulate and activate non-neuronal cells, including Schwann cells and perineurial cells, followed by axonal growth that is supported by these activated non-neuronal cells (Toews et al., 1998). Further, rapid invasion of macrophages enhances phagocytotic activity to dispose of cell debris (Hall, 2001). In the adult mammalian CNS, on the other hand, macrophages invade very slowly and poorly (Lazarov-Spiegler et al., 1998). The importance of macrophage invasion is implied from the observation that the transplantation of macrophages stimulates a regenerative response in the transected rat optic nerve (Schwartz et al., 1999). Despite repulsive glial characters, recent studies revealed that CNS axons show a regenerative response after artificial manipulations of their surrounding microenvironments (Fouad et al., 2001; Jones et al., 2001). The failure to regenerate is not simply an intrinsic deficit of CNS neurons, and is blocked by the CNS environment. A feasible strategy for augmenting CNS regeneration is to adopt the favorable properties exhibited in peripheral nerves after injury, specifically, the provision of trophic factors and the grafting of Schwann cells to promote axonal regeneration. A series of experiments performed in the past decade have demonstrated that implantation of a peripheral nerve segment into the transected optic nerve promotes the regeneration of nerve fibers for distances of several centimeters into the implanted tissue (Vidal-Sanz et al., 1991), and that the peripheral nerve graft can delay nerve cell death and prevent axonal degeneration (VillegasPerez et al., 1988). It must be emphasized that the peripheral nerve segment transplanted in the CNS should be in the process of Wallerian degeneration,

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Nerve regrowth by cell transplantation and therefore contain activated Schwann cells within the degenerating segment. These Schwann cells provide suitable substrates that are critical for the regeneration of CNS axons. In the case of the optic nerve, direct contact of optic nerve axons with living Schwann cell seems to be more effective for successful regeneration rather than supplying trophic factors (Dezawa & Nagano, 1993; Dezawa et al., 1997); however, it should not directly exclude the substantial role of trophic factors and extracellular matrix during regeneration as these elements are not sufficient in supporting longlasting regeneration of CNS axons in vivo. The CNS regeneration can be sustained when the relationship between axons and viable Schwann cells is closely and intimately maintained (Dezawa & Nagano, 1993; Dezawa et al., 1997). It would be more adequate to say that direct contact with Schwann cells initially provides a cellular foothold, and then supplies growth factors and extracellular matrix molecules to the regenerating CNS axons. The intimate relationship of axon and the Schwann cell based on structural and molecular linkages is discussed in the following section.

Axon–Schwann cell interaction during sciatic and optic nerve regeneration Together with production of neurotrophic factors and extracellular matrix molecules that support neuronal survival and axonal growth, Schwann cells express a wide range of cell adhesion molecules such as the immunoglobulin superfamily, cadherins and integrins (Martini, 1994; Ide, 1996). Some of these molecules are also found in growth cones, and known to operate the interaction between these cells (Ide, 1996). Above all, NCAM, L1 and N-cadherin were found to be closely related to PNS and CNS regeneration because these molecules were widely detected at the interface of axon and Schwann cell for a longer period during regeneration (Martini, 1994; Ide, 1996). Extracellular molecules, particularly laminin, merosin and fibronectin, are known to stimulate neurite outgrowth (Agius & Cochard, 1998). The cellular counterpart of extracellular matrix, a-1-b-1 or a-6-b-1 integrin subunit, is reported to mediate the attachment between axon and Schwann cell basement membranes in neurite outgrowth (Ide, 1996). The importance of MAG in axon–Schwann cell communication was indicated by MAG-deficient mice in which axonal loss and demyelination occur later in the life (Carenini et al., 1997). It is believed that MAG is a modulator of axonal properties that regulate a kinase-phosphatase system within the axon, and is also related to the process of

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remyelination of regenerating nerve fibers by Schwann cells (de Waegh et al., 1992). First, it is rarely detectable at the interface between unmyelinated axons and Schwann cells, but as myelination becomes advanced by Schwann cells, MAG increases and is present at the periaxonal surface (Dezawa & Nagano, 1996). This result suggests that an intricate temporal and spatial regulation of MAG expression exists during the process of remyelination. Recent studies demonstrated that neurite extension stimulated by cell adhesion molecules does not rely simply on adhesion, but instead requires that second messenger cascades in neurons is activated, thereby inducing axonal elongation (Doherty & Walsh, 1994). Detailed observations of the mutual functions between nerve axons and Schwann cells in sciatic and optic nerve regeneration revealed that both cells are not simply connected via cell adhesion molecules, but they seem to have a closer structural and functional relationship via tight and gap junctions. Usually, these connecting structures are formed between homologous cell types, but the possibility exists of a temporary development of typical tight and gap junctions between heterologous tissues of the nerve axon and Schwann cells during regeneration. Mature tight junctions in the intestine and other epithelial tissues show a belt-like meshwork in the freeze-fracture image that provides a barrier to the free passage of molecules and ions across paracellular pathways (Farquhar & Palade, 1963). However, tight junctions observed between the regenerating axons and Schwann cells were short focal fusions of the outer leaflets of two adjoining plasma membranes (100–500 nm in length), and were mostly isolated (Dezawa & Nagano, 1993; Dezawa et al., 1996). Such a structure seems to serve as a site of adhesion and provides mechanical links for regenerating axons to facilitate stable interactions with Schwann cells, rather than sealing structures (Dezawa & Adachi-Usami, 2000). There have been several reports of gap junctions with connexin 32 in nervous tissues, such as brain and peripheral nerves, but all were between homologous cell types, for example, Schwann cell processes of the Schmidt–Lanterman incisures and paranodal portions of the nodes of Ranvier, and between adjoining Schwann cells in regenerating peripheral nerves (Dermietzel & Spray, 1993; Chandross, 1998). They have also been found between the immature and undifferentiated cells in early developing embryos (Tetzlaff, 1982; Warner, 1992). The gap junction between heterologous cell types of regenerating axon and Schwann cells was mostly a small plaque that consisted of several intramembrane

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Figure 1. Schematic summary of axon–Schwann cell interaction during regeneration of peripheral nervous system (PNS) and central nervous system (CNS).

particles, associated with either connexin 32 or connexin 43 immunoreactivities (Dezawa et al., 1998; Dezawa & Adachi-Usami, 2000). Intercellular coupling between the regenerating axon and Schwann cell pair was confirmed by intracellular injection of biocytin (a membrane-impermeable dye of low molecular weight of 373 Da). The axon and the slender process of an adjacent Schwann cell are labeled with biocytin, but the cytoplasm of other Schwann cells nearby are not (Dezawa et al., 1998; Dezawa & Adachi-Usami, 2000). The result implied that direct inter-communnication via gap junctions exists between axon and Schwann cells during nerve regeneration through which nutrients and cytoplasmic factors can pass back and forth. Cell-adhesion molecules and neurotrophic factors, in most cases, act via membrane receptors. In contrast, the existence of gap junctions suggests that these structures might provide a ‘hot line’ for the direct trafficking of small but significant quantities of essential intracellular factors between axons and Schwann cells in the process of regeneration. In conclusion, Schwann cells contribute to nerve regeneration by producing trophic factors and celladhesion molecules, in addition to providing junctional structures to stabilize cell contact and facilitate the traffic of substances between the regenerating axon and the Schwann cell. The schematic summary of axon–Schwann cell interaction during nerve regeneration is shown in Fig. 1.

Reconstruction of optic nerve circuit by Schwann cell transplantation Some kinds of cells, such as gene-transferred astrocytes, amniotic cells, olfactory ensheathing cells and neuronal stem cells, can induce the elongation of CNS nerve fibers (Bankiewicz et al., 1994; Castillo et al.,

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1994; Lundberg et al., 1997; Ramon-Cueto et al., 1998). However, we must consider that many CNS axons are myelinated by oligodendrocytes. The optic nerve tract is one of the typical examples. Myelination is known to control the number of neurofilaments and elevate the phosphorylation state of neurofilaments in the axon, eventually leading to the large axon caliber. Conversely, absence of myelin results in lower amounts of neurofilaments, reduced phosphorylation levels and smaller axon diameter (Martini, 2001). In addition, myelinating Schwann cells and oligodendrocytes mediate the spacing of sodium channel clusters during development of the node of Ranvier (Martini, 2001). Therefore, even if the CNS can elongate their axons, remyelination of regenerated axon is indispensable for the re-establishment of CNS function. Although Schwann cells originally myelinate peripheral axons, they can also remyelinate CNS axons when transplanted. Accordingly, they are purposive cells and are one of the best candidates for implantation methods for the regeneration of the CNS. After a peripheral nerve bridge is inserted between the severed optic nerve and the superior colliculus, regenerating RGC axons grow through the bridge, then extend, arbolize and form synapses in the superior colliculus that persists for long periods of time (VidalSanz et al., 1991). The ratio of regenerating axons, however, represents less than 10% of the total population (Vidal-Sanz et al., 1991). Considering the evidence that the intimate and direct interaction is maintained between the regenerating axon and the Schwann cell, the increased process of regeneration will be achieved by the increase in opportunities in encounterering axons with Schwann cells. To obtain a fully functional recovery of transected optic nerve fibers, new artificial grafts consisting of high-density cultured Schwann cells were designed. Cultured Schwann cells were purified from dorsal root ganglions of newborn rats (Wistar strain), and approximately 106 cells/mL were suspended in an extracellular matrix and trophic factors of nerve growth factor (NGF) 100 ng/mL and bFGF (100 ng/mL), so that Schwann cells in the tube were approximately 100 times as dense as is normal in the PNS. They were transferred into 1.5–2 cm in length of permeable tubing (Negishi et al., 2001). Among the interventions tested to improve the axonal regeneration achieved with Schwann cell transplantation is the addition of neurotrophic factors. In the preliminary experiment, NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-4/5 and bFGF were supplied to the artificial graft segment (1 cm in length) and the ratio of regenerated RGCs were evaluated after the transplantation, revealing that

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Nerve regrowth by cell transplantation combined supply of NGF and bFGF are more effective. Accordingly, they are supplied to the artificial graft as noted above. These factors might activate and prolong the survival of Schwann cells, and might also act on damaged RGCs (Yip & So, 2000). In rats, almost all optic nerves project to the contralateral side of the superior colliculus. Normally, optic nerve fibers run at the bottom of a skull, but in the experimental condition, the left optic nerve fibers were dissected just behind the eyeball, and they were bridged with an artificial graft lying on the upper surface of a head to the right superior colliculus. Soon after the transplantation, BDNF (100 ng/mL), CNTF (50 ng/mL) and forskolin (FSK) (5 mmol) were injected into the vitreous body. The supply of neurotrophins together with FSK are reported to be efficient for the survival of RGCs (Meyer-Franke et al., 1998). Forskolin increases the level of intracellular cyclic adenosine monophosphate (cAMP). Such an increase of intracellular cAMP leads to the enhancement of the responsiveness of cells to trophic factors by quickly translocating the trk receptors stored in an intracellular synaptic vesicle-like organelle to the plasma membrane of the CNS (Meyer-Franke et al., 1998).

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As RGCs are known to respond to BDNF, the addition of FSK will be expected to enhance the survival of RGCs after intravitreous injection. Moreover, CNTF are reported to be sufficient for the axonal regrowth of RGCs, rather than the enhancement of survival (Cho et al., 1999). From this evidence, the intravitreous injection of BDNF and FSK with CNTF was performed. The optic nerve fibers successfully regenerated within the artificial graft for a few centimeters to the CNS target of the superior colliculus (Fig. 2). To estimate the regeneration of optic nerve fibers, diI 1, 1′-dioctadecyl3, 3, 3′, 3′-tetramethylind carbocyanine percholorate retrograde labeling, cholera toxin B subunit anterograde labeling, immunohistochemistry and electron microscopy were performed. At 7.5 months after transplantation, parenchymatous tissue was observed within the tube. Intravitreous injection of cholera toxin B subunit labels RGC axons anterogradely, and the detection of cholera toxin B subunit by immunohistochemistry enables us to distinguish the regenerating RGC axons from other neuronal axons. The penetrtion of anterograde-labeled RGC axons were abundantly recognized within the superior colliculus, and were mostly positive for synaptophysin, indicating that

Figure 2. Optic nerve regeneration by artificial Schwann-cell graft. The left optic nerve was dissected just behind the eyeball, and was bridged with an artificial graft lying on the upper surface of a head to the right superior colliculus. (a) Anterograde labeling of regenerated optic-nerve fibers by cholera toxin B subunit. At 7.5 months after the operation, many labeled (green) fibers were observed within the superior colliculus. Synaptophysin reactivity (red) is colocalized with labeled axons. (b) Retrograde labeling of retinal ganglion cells (RGCs) by diI. Labeled ganglion cells were detected in the whole mount retina. (Dezawa, in press. Reproduced with permission from Molecular Medicine)

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regenerated optic nerve fibers participated in the reformation of synapses in the superior colliculus (Fig. 2a). DiI retrograde labeling (injected into the superior colliculus) showed that approximately 18% of total RGC axons regenerated into the superior colliculus (Fig. 2b). Axons within the artificial graft tube were mostly ensheathed by Schwann cells (Negishi et al., 2001). Immunoelectronmicroscopy revealed that some of the neurites labeled with cholera toxin B subunit were myelinated by Schwann cells (Negishi et al., 2001). In order to evaluate the function of regenerated optic nerve fibers, a visual prepulse task was used to estimate the recovery of visual function in the peripheral nerve grafted animals (Sasaki et al., 1998). A sudden intense noise is known to cause a startle response in many animals. A brief stimulus of flashlight (prepulse), which in itself does not cause the startle response, preceding the acoustic startle stimulus (white noise) with lead times of 50–400 msec, inhibits the startle response. Amplitudes of startle movements were measured by a piezoelectric accelerometer (YGH313AGA-245SO; Keyence, Osaka, Japan) that was attached to the center at the bottom of the chamber. Startle amplitude was measured as a maximal peak amplitude of the wave output from the sensor that occurred between the onset of startle stimulus and 200 msec after the stimulus, and amplitude with or without prepulse was compared and statistically analyzed (Fig. 3). Such an effect is called prepulse inhibition, and has been reported to be useful for studying the ability to detect visual signals in animals (Sasaki et al., 1998). Prior to the task, the optic nerve fibers of the contralateral intact side were severed so that the visual information was sent to the CNS only by the optic nerve fibers via the implanted graft. Accordingly, if the startle amplitude was significantly decreased by the prepulse flashlight, functional recovery seems to occur in the retino-tectal pathway

after the artificial Schwann cell graft that bridges the cut end of the optic nerve and the superior colliculus. Behavioral experiments were conducted 7.5 months after implementation. Two different stimulating sets were prepared, the first was noise only, the second was flashlight (prepulse) followed by noise. Each rat was first exposed to 10 trials by the first set, then the rat was randomly exposed to the first and/or second stimulating sets 20 times each. Two out of the nine rats showed a decrease in startle amplitudes (8.4–15.8%) with the preceding flashlight. Although additional months might be necessary for the conspicuous recovery of visual function, the fact that depression was observed is noteworthy because it was a successful sign of recovery in visual function in the implanted rats. Further detailed and long-term experiments are required in the future. In the past, it was assumed that optic nerve fibers had no ability to regenerate neurites under any circumstances. At present, it has been shown that optic nerve fibers can regenerate their neurites for up to several centimeters under the proper circumstances, such as an artificial environment of Schwann cells. Furthermore, the visual function recovers again if the regenerating optic nerve fibers extended to the superior colliculus, thereby making functional connections.

Sciatic and optic nerve regenration by transplantation of transdifferentiated bone marrow stromal cells In addition to stem cells for hematopoietic cells, bone marrow contains stem-like cells that are precursors of non-hematopoietic cells and can be cultivated in vitro. They were initially referred to as plastic adherent cells or colony-forming-unit fibroblasts and recently as mesenchymal stem cells or marrow stromal cells (Prockop, 1997). Marrow stromal cells are connective

Figure 3. Diagrammatic summary of the visual prepulse experiment.

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Figure 4. Immunocytochemistry of p75 (a,e), S-100 (b,f), glial fibrillary acidic protein (c,g) and O4 (d,h) in differentiated marrow stromal cells (a–d) and Schwann cells (e–h). Scale bar = 50 mm. (Dezawa, in press. Reproduced with permission from Molecular Medicine)

tissue elements that provide structural and functional support for hemopoiesis, but they are now recognized as progenitor cells capable of differentiating into mesenchymal tissues of bone, cartilage, fat, muscle and other connective tissue (Prockop, 1997). In addition to these tissues, transdifferentiation of MSCs into the neuronal lineage such as astrocytes and neurons has been reported (Sanchez-Ramos et al., 2000; Woodbury et al., 2000; Deng et al., 2001). Therefore, they are an interesting target for use in neural transplantation and regeneration. This section demonstrates that MSCs can be induced to differentiate into cells with Schwann cell characteristics that are capable of eliciting regeneration of the sciatic and optic nerves. MSCs were obtained from adult rat (Wistar strain) bone marrow, and were subcultured four times. They were then treated with b-mercaptoethanol (BME) followed by retinoic acid (RA) and cultured in the presence of FSK, bFGF, PDGF and heregulin (HRG) (Dezawa et al., 2001). Such differentiated MSCs were morphologically different from the original undifferentiated MSCs and resembled primary cultured Schwann cells (Dezawa et al., 2001). To evaluate the nature of the differentiated MSCs, immunocytochemistry of p75, glial fibrillary acidic protein (GFAP), S-100 and O4, all known as markers of Schwann cells (Shah et al., 1996; Jessen & Mirsky, 1999; Morrison et al., 1999), was performed and the results compared with those obtained from Schwann cells. Most differentiated MSCs and Schwann cells were positive to p75 and S-100, and some differ-

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entiated MSCs and Schwann cells were positive to GFAP and O4 (Fig. 4). Undifferentiated MSCs were not positive to p75, GFAP and O4, whereas a few cells were positive to S-100. Consequently, the results indicated that cells with a phenotype similar to that of Schwann cells could be induced from the original MSCs by the treatments. Prior to transplantation, MSCs were infected with retrovirus to introduce green fluorescent protein (GFP) genes, allowing distinction from host cells. MSCs were then differentiated as described above, suspended in Matrigel (Collaborative Biomedical Products, Bedford, MA) at a concentration of 1–2 × 107 cells/mL and were put into the permeable tube (Amicon, Beverly, MA). This type of MSC graft (approximately 1.5 cm in length) was anastomosed to the cut end (proximal side) of the adult rat (Wistar strain) left sciatic nerves, and the distal end of the graft was plugged with surgical adhesive (Sankyo, Tokyo, Japan; Dezawa, in press). Three weeks after the operation, signals to GFP, GAP-43 and myelin basic protein (MBP) were all detected in the graft, showing that the regrowth of both nerve fibers and differentiated MSCs existed and were accompanied by myelination (Fig. 5a–c). Myelination detected by the MAG signal was colocalized with the GFP signal, recognized, shown as light blue color-coding in Fig. 5d. Nodes of Ranvier can also be recognized in some areas (Fig. 5e). The graft of differentiated MSCs was also examined by immunoelectronmicroscopy. Myelinated axons were recognized within the graft, and GFP-signals were

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Figure 5. (a–e) Laser confocal microscopy of artificial marrow stromal cell (MSC) grafts transplanted to the cut end of the sciatic nerve 3 weeks after the operation. Differentiated marrow stromal cells with green fluorescent protein (GFP) expression are seen within the artificial tube. (a) growth associated protein-43 positive nerve fibers penetrated into the graft (b) and myelin basic protein (c) are detectable within the graft. At the upper edge of part a, part of the hollow fiber is seen. The proximal nerve segment is situated on the left side, and the distal end of the artificial graft is on the right side. Scale bar = 500 mm. Parts d and e show the immunohistochemistry of myelin associated protein (MAG) (blue), and neurofilament (red) of the differentiated MSC graft. (d) MAG-positive myelin composed of GFP-delivered MSCs (blue, arrowheads). (e) MAG and neurofilament staining, with Ranvier’s nodes indicated by arrowheads. Scale bar = 20 mm. (f ) Immunoelectron microscopy of differentiated MSC graft 3 weeks after operation. Axon (Ax) and myelinating MSC (M) are shown. The GFP signal is localized in myelin and cell membranes of the MSCs. Basal laminae are indicated by an arrowhead. Scale bar = 1 mm. (Dezawa et al., 2001. Reproduced with permission from Molecular Medicine)

clearly seen on the myelin and occasionally on the cell membrane of the myelin-forming cells (Fig. 5f). These results indicated that MSCs were directly associated with the myelin formation of regenerating nerve fibers within the graft. The MSC graft was also transplanted to the cut end of adult rat optic nerve, and the nerve regeneration was evaluated. Prior to transplantation, differentiated MSCs were labeled with Hoechst 33342 (Sigma, St Louis, MO). The graft (approximately 1.5 cm in length) was prepared as described above, anastomosed to the cut end of the optic nerve and the distal end of the graft was plugged with surgical adhesives. Regenerating optic nerve fibers were abundantly observed within the graft, and in most cases were in contact with Hoechst-labeled differentiated MSCs (Fig. 6). The expression of myelin protein

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MAG was detected in these MSCs, as in case of sciatic nerve regeneration. In the implants of undifferentiated MSCs, although the regeneration was observed, the number of regenerating neurites was smaller and the length of elongation was significantly shorter than that of differentiated MSCs both in the sciatic (data not shown) and optic nerve (Fig. 5b) regeneration. With the differentiated MSC graft, the distance of sciatic nerve regrowth reached 8–10 mm at 3 weeks, while with the undifferentiated MSC graft, a growth of only 2.5 mm was achieved over the same time. Further, neither the formation of myelin nor of Ranvier’s nodes were observed with the undifferentiated MSC graft. This suggests that differentiated MSCs are more useful for the successful regeneration of nerve fibers. The reason why undifferentiated MSCs induce nerve fiber

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Figure 6. Artificial marrow stromal cell (MSC) grafts transplanted to the cut end of the optic nerve 10 days after the operation. (a) Differentiated MSCs were transplanted and many growth associated protein 43 (GAP-43) immunoreactive nerve fibers (green) were seen within the graft. (b) Transplantation of undifferentiated MSCs resulted in the small number of GAP-43 positive nerve fibers. Approximate position of the transitional zone of the optic nerve and graft is indicated by arrows in parts a and b. (c,d) Higher magnification of part a. Most of the GAP-43 positive fibers were in contact with Hoechst-labeled differentiated MSCs. Scale bars; b = 300 mm, c and d = 50 mm (Dezawa, in press. Reproduced with permission from Molecular Medicine).

regrowth, even though the regeneration was less vigorous, might be attributed to the production of cytokines by MSCs. For example, MSCs produce IL-6, which is known to act as a trophic factor for regenerating nerve fibers (Gimble et al., 1991; Shuto et al., 2001). On the basis of our results, the sequential administration of various factors of BME and RA followed by a mixture of FSK, bFGF, PDGF and HRG effectively induces the differentiation of MSCs into myelinating Schwann cells, thus assisting the regeneration of nerve fibers. b-mercaptoethanol is a reducing agent, and is known to induce morphological changes when administrated to MSCs (Woodbury et al., 2000; Deng et al., 2001), while RA is a morphogen, and was reported to induce differentiation of embryonic stem (ES) and neural stem cells into nerve cells by regulating the expression of various transcription factors that are crucial to an early neuronal determination such as MASH1 and NeuroD (Takahashi et al., 1999). Accordingly, BME and RA are presumed to work as triggering factors, inducing changes in the morphological and transcriptional characteristics of MSCs. Basic fibroblast growth factor, PDGF and HRG are effective in the differentiation and proliferation of glial and Schwann cells (Jessen & Mirsky, 1999). Particularly, the subtype of neuregulin, HRG, instructively influences the decision of cell fates and was reported to induce neural crest cells to develop selectively into Schwann cells (Shah et al., 1996). Heregulin also activates phosphatigylionsitol-3-kinase in Schwann cells, which promotes the early stages of myelination during development (Maurel & Salzer, 2000).

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As noted above, FSK increases the level of cAMP, which increases mitogenic responses at an early time perhaps by stimulating the expression of growth factor receptors (Meyer-Franke et al., 1998). Thus, FSK together with bFGF, PDGF and HRG could have a synergistic effect in enhancing these factors to MSCs. Although further investigations on the mechanism of the MSC differentiation are required, the sequential administration of the reagents used in this experiment seems cumulative for the differentiation of MSCs to express the Schwann cell phenotype because an omission of any one of the above factors resulted in an incomplete differentiation of MSCs regarding morphology and immunoreactivity to Schwann cell markers.

Conclusion In mammals, the damaged CNS does not regenerate. In contrast, peripheral nerve fibers have the capability to regenerate. When injured, anastomosis of transected segments allows the PNS axons to elongate through the distal segment and re-establish synaptic connections with the targets, and ultimately recover normal function. Activated Schwann cells, under Wallerian degeneration, play an important role in the series of reactions. Despite the fact that both the glial cell of astrocytes and oligodendrocytes and Schwann cells are constituent members of nerve tissues, they have very different functions in regeneration. There are some qualitative differences between them. First, during development,

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glial cells in the CNS derive from the neural tube, whereas Schwann cells derive from the neural crest (Jessen & Mirsky, 1999). Thus, they are originally derived from different precursors. Second, astrocytes and oligodendrocytes contrast sharply with Schwann cells. That is, they neither return to be an immature phenotye nor do they alter their character even when the CNS is injured. Instead, they keep their highly differentiated structure as in normal circumstances. On the contrary, Schwann cells, though they are already fully differentiated and extending myelin sheaths, rapidly change their structure and characteristics after injury to become undifferentiated, and then follow active mitosis and proliferation. Unlike astrocytes and oligodendrocytes, Schwann cells are more flexible in nature and can rapidly adopt to emergencies, including nerve injury. Along with the well-known functions of the Schwann cell such as the production of trophic factors and cell adhesion molecules, Schwann cells contribute to nerve regeneration by providing tight junctions to stabilize cell contact with injured nerve cells, as well as gap junctions to facilitate traffic of substances between the cells (Dezawa & AdachiUsami, 2000). Previously, the tight and gap junctions were supposed to exist only in homologous cells. However, results show that both structures can be present between heterogeneous cells such as the Schwann cell and nerve axons in exceptional cases such as nerve regeneration. The detailed functional mechanisms of the junctions should be elucidated, but at present, it is presumed that this structure might act as a foothold in the tissue where the regeneration and remodeling of cells occur. If the overall goal of CNS reconstruction is initially the survival and maintenance of nerve-cell bodies followed by the elongation of nerve fibers and finally the reconstruction of neural projection and functional recovery, then we must say that our goals are still distant. Cell transplantation to repair CNS injury is a vibrant area of research where the goal is to alleviate functional deficits. Recent studies indicate that implantation of Schwann cells as well as some other kinds of cells, such as gene-transferred astrocytes, amniotic cells, olfactory ensheathing glial cells, ependymal cells, differentiated ES cells and neural stem cells, can also induce neurite elongation in the CNS (Bankiewicz et al., 1994; Castillo et al., 1994; Lundberg et al., 1997; Ramon-Cueto et al., 1998; Johansson et al., 1999; Ide et al., 2001; Sawamoto et al., 2001). However, considering that almost all of the intact CNS fibers, such as optic nerve fibers, are myelinated fibers, complete functional recovery might not be achieved without their remyelination. Fortunately, Schwann cells can induce the elongation of optic nerves as well as

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remyelination of the elongated RGC axons. Accordingly, they are good substrate in the implantation methods for regeneration. Marrow stromal cells are certainly one of the most attractive cells for an application of neural transplantation because they exhibit several important and potential advantageous features both in PNS and CNS regeneration. In addition, they are easy to isolate from bone marrow aspirates in the clinic and can readily be expanded under culture conditions for autologous transplantation. Therefore, differentiated MSCs are considered by us to become one of the strongest candidates for cell transplantation involving tissues of the nervous system. In the near future, success in functional regeneration of CNS can be expected, particularly in light of further progress made in related fields.

Acknowledgements I wish to acknowledge the excellent support of Dr Masahiko Takano, Department of Ophthalmology, Yokohama City University School of Medicine, for the optic nerve transplantation, and Drs Hitoshi Sasaki, Hajime Sawai and Yutaka Fukuda, Department of Physiology, Osaka University Graduate School of Medicine, for the visual prepulse task experiment. I also wish to thank Professor Hajime Sawada, Department of Anatomy, Yokohama City University School of Medicine, and Professor Emeritus Toshio Nagano, Department of Anatomy, Chiba University School of Medicine, for their support and encouragement.

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