Abstract
Over the past decade, olfactory ensheathing cell (OEC) transplantation has emerged as a promising treatment for spinal cord injury. OECs support the continuous neuronal turn-over of the olfactory system. Transplants of these cells into rodents with spinal cord injury (SCI) has promoted axonal regeneration and partially restored motor function. There is currently much debate over whether OEC transplants are ready for clinical trials. This paper reviews what is and is not known about OECs. It argues that we must learn more from further preclinical reseach before transplanting OECs into human SCI.
The treatment of spinal cord injury (SCI) has received a lot of attention in neuroscience research. Although the peripheral nervous system shows great capacity for repairing transected axons, the same damage to spinal cord neurons leads to a devastating, permanent loss of function (Ramon y Cajal, 1928). The lack of axonal regeneration in the central nervous system (CNS) is not simply caused by the inherent properties of the neurons themselves, but is due to inhibition from other cells in their environment. Oligodendrocytes and astrocytes are major sources of inhibitory signals, such as MAG, proteoglycans, and versican (McKerracher, 2001). Much research is dedicated to replacing the unfavorable environment of the injured spinal cord with one that facilitates axonal regeneration and target innervation. Transplants have shown promising preclinical results, but there are disadvantages of each transplanted cell type. For example, Schwann cells (SCs) release several neurotrophic factors and adhesion molecules that support axonal survival and regrowth (Tuszynski et al., 1998). However, the effect of transplanted SCs on axonal regeneration is often limited to the grafted area (Xu et al., 1997). That is, the growing axons do not exit the graft into the less favorable spinal cord tissue, preventing them from extending all the way to their intended targets.
The best cell candidate for spinal cord transplantation may be olfactory ensheathing cells (OECs). Like SCs, OECs support axonal regeneration and remyelination of CNS neurons. But OECs show an important advantage over SCs: the former freely migrate into CNS environments and function normally among astrocytes (Lakatos et al., 2000). Pre-clinical trials of OEC transplants in SCI have exploded over the past decade. As hoped, OECs have improved CNS repair in vitro and in vivo. Some people are very excited about the potential benefits of OEC autotransplantation, and are pushing to get clinical trials underway. Others are skeptical of beginning clinical trials.
A debate has arisen: Is it reckless to begin human trials with these mysterious cells, or is it inhumane to deprive SCI patients of this possibly beneficial treatment? This paper will review what is and is not known about the biology of OECs. It will then briefly review the preclinical studies of OEC transplantation in SCI. Finally, it will argue that our current understanding of OECs is not sufficient to begin clinical trials. There are many important unanswered questions concerning the physiology and therapeutic benefits of OECs (especially human OECs), so transplantation into the injured human spinal cord may be ineffective and dangerous. Unsuccessful clinical trials could diminish hope and scientific interest in OECs, thereby dismissing a potentially remarkable cure.
The olfactory nervous system has an ability for regrowth that is uncharacteristic of other CNS areas. Throughout an animal's life, olfactory neurons constantly die and are replaced by neurons from the basal stem cell layer of the olfactory epithelium. The growing axons of new olfactory neurons reestablish the connections between the nose and brain, and the cycle repeats every 30 to 60 days (Kandel et al., 2000). OECs are glia that are found along the PNS-CNS border, from the olfactory epithelium to the nerve fiber layer of the olfactory bulb (see Figure 1). OECs operate in both of these environments to support the ongoing axonal growth, ensheathment, and targeting of olfactory neurons (Franklin and Barnett, 2000). OECs use cytoplasmic extensions to ensheath groups of axons (Barber and Lindsay, 1982). They also migrate ahead of growing axons, guiding and supporting their growth (Tennet and Chuah, 1996). Relatively little is known about the cellular mechanisms of OECs, but investigations into their
immunocytocemistry are offering insights into their physiological capabilities.
OECs show a complex heterogeneity of immunocytochemical properties, that varies across animal ages, species, locations in the olfactory system, and in vitro culture media (Chauah and West, 2002, and Moreno-Flores et al., 2002). For simplification, OECs are usually described in terms of their two most common phenotypes: Schwann-like and astrocyte-like (see reviews by Bartolomei and Greer, 2000 and Lu and Ashwell, 2002). The astrocyte-like phenotype dominates OECs in the olfactory bulb. These cells express glial fibrillary acidic protein, are immunoreactive for Rat-401, form end-feet junctions at blood vessels, and form the glial limitans of the olfactory system. The Schwann-like phenotype is more common in the peripheral olfactory system and in vitro, and this variety is thought to be responsible for remyelinating axons and supporting axonal growth. The Schwann-like behavior of OECs is correlated with their Schwann-like immunoreactivity for markers such as p75NGFR (low affinity NGF receptor), L1 (cell adhesio molecule), A5E3 (vimentin, a neurofilament), and Po (a Schwann cell specific marker). More detailed descriptions of OEC immunocytochemistry in vivo and in vitro are provided in Tables 1 and 2. The morphology of OECs are spindly-bipolar (Schwann-like) or flat-angular (astrocyte-like), (Barber and Lindsay, 1982).
The actual diversity in OEC phenotypes is much more complicated than the above dichotomy implies. OECs show various combinations of immunoreactivity, morphology, and behavior. However, all these phenotypes are thought to develop from a common epithelial cell progenitor (Chuah and Au, 1991). Future research should aim to expand upon our current understanding of OEC phenotypes through further immunoreactive, morphological, and behavioral characterization. By understanding the factors that determine OEC phenotypic expression we can improve our ability to utilize these cells in spinal cord therapy. We will be able to culture OECs in media that activate expression of the most appropriate phenotypes for axonal regeneration.
The ability of the primary olfactory system to continuously turn-over its neural population has made its unique glia a prime candidate for CNS transplantation. OECs have been shown to repair neuronal damage in olfactory bulbs (Kott et al., 1994), the fimbria-fornex (Smale et al., 1996), and the thalamus (Perez-Bouza et al., 1998). The first test of OEC transplants in SCI was very successful (Ramon-Cueto and Nieto-Sampedro, 1994). In this experiment, transplanted OECs promoted regeneration of transected dorsal root axons in rats. OECs accompanied axons as they passed by the glial scar, into the dorsal horn (see Figure 2). This was an improvement over SC transplants because OECs could freely migrate into white matter, gray matter, connective tissue, and glia to support axonal regeneration over long distances in the spinal cord (Ramon-Cueto et al., 1998). Moreover, the regenerating axons tended to innervate the correct spinal cord laminae. These exciting findings led to many other studies of OEC transplantation,and the results continued to be encouraging. Li et al. (1997, 1998) were among the first to show that OEC transplants could restore behavioral by regenerating spinal cord axons, and others have similarly observed improvement in coordinated locomotion, weight-bearing, touch, and proprioception following this treatment (Ramon-Cueto et al., 2000). A recent experiment showed that OECs promote axonal regeneration after delayed transplantation of up to four weeks after SCI (Lu et al., 2002). In addition to supporting axonal regeneration, OECs are able to remyelinate axons in the spine (Franklin et al., 1996), thereby improving action potential conduction in neurons (Imaizumi et al., 1998).
As convincing as the above evidence may be, there are many important things that we must learn about OECs before we can be confident of their success in human SCI. Perhaps the most difficult questions are about the very nature of OEC function: What mechanisms underlie their ability to repair CNS damage? There are many characteristics of OECs that could improve axonal regeneration. Three main explanations are currently being proposed. First,they produce and release several neural growth factors, including FGF1 (Key et al., 1996), FGF2 (Chuah and Teague, 1999), NGF, BDNF, GDNF (Woodhall et al., 2000), and GGF2 (Chuah et al., 2000). Growth factors improve the survival of neurons, and signal axonal growth by mechanisms that are just beginning to be understood. For example, priming spinal neurons with BDNF, GDNF, or NGF elevates cAMP and activates PKA, by blocking inhibition from MAG and myelin (Cai et al., 1999). OECs also express NGFr, GFRa-1 and GFRa-2, which are the receptors for NGF, GDNF, and NTN,
respectivly (Woodhall et al., 2001). In particular, OECs express TrkB, but not TrkA or TrkC versions of the NGFr. Similarly, although they express GFRs, they do not produce RET. TrkA and RET are necessary to activate intracellular signaling from these growth factors, so OEC receptors probably bind their ligands to present them to other olfactory cells (Chuah and West, 2002). Secondly, OECs express laminin, an extracellular matrix molecule (Treloar et al., 1996), which promotes extension of neurites, and may be involved in migration of OEC (Tisay and Key, 1999). Third, they release two cell adhesion molecules: N-CAM (Miragall et al., 1988) and L1 (Kafitz and Greer, 1997), which support axonal elongation. OECs further assist axonal growth by secreting Semaphorin-3A, a chemorepulsion molecule that guides growing axons to their targets (Pasterkamp et al., 1998). We do not know how the above proteins interact with each other and with growing axons. Future in vitro studies should investigate the functions of OEC
proteins by using antibodies to bind receptors, or antisense inhibition to downregulate protein expression. In situ hybridization studies would allow us to identify which proteins are primarily translated by OECs after transplantation into the spinal cord. These experiments may help us understand the mechanisms by which OECs promote axonal regeneration.
Once we have identified the OEC properties responsible for axonal regeneration, we must strive to understand how these properties are expressed across the range of OEC phenotypes. For example, do different phenotypes make different contributions to the neurotrophin cocktail in OEC populations, or are there neurotrophins that are common to all transplanted OECs? In addressing these questions, it is important to study human OECs, which may be quite different from those of the rat. Studies of human OECs are very few, perhaps because they are very difficult to grow in vitro (Lu and Ashwell, 2002). Nevertheless, some groups have been able to cultivate OECs from human olfactory bulbs, and have transplanted them into rodents with demyelinated spinal cords. In these experiments, the human OECs were successful in remyelinating dorsal root axons (Barnett et al., 2000; Kato et al., 2000). There is only one preclinical study of human OEC transplantation in SCI. The transplants improved axonal regrowth and several rats
even recovered some walking ability (Bernal et al., 2002). These exciting studies give us great hope in spinal cord repair, and help to raise public awareness and research funding.
A major concern in using human OEC transplantation is finding an accessible source of cells. The olfactory bulbs and epithelium of rodents are proportionally much bigger than those of people. Biopsied human OECs must be grown in vitro to obtain an adequately large population of cells for transplantation into the spinal cord. This warrants research into culturing and cloning OECs. Furthermore, all the past studies of human OEC transplantation have obtained cells from the olfactory bulbs. One of the great clinical appeals of OEC transplantation is the easily accessible source of OECs in the olfactory epithelium. A bulbectomy may severely disrupt the patient's sense of smell, but samples of the olfactory epithelium may be removed without behavioral consequences (see Franklin, 2002). We know that separate OEC phenotypes are concentrated in the central and peripheral olfactory system, so transplants of human OECs from the olfactory epithelium need to be tested in rodent models of SCI. This will show that OECs are not only beneficial, but are easily accessible for autotransplantation.
The OEC literature has another crucial gap: the rat models of SCI consist of complete cuts or lesions of the spinal cord, which does not resemble the SCIs of most people. In real life, SCI usually involves a contusive injury, maintaining a proportion of white matter across the lesion (Kakulas, 1999). Treatments that repair a transected spinal cord may be less effective in a contusive injury, because of differences in inflammatory responses and neuronal damage (Tuszynski and Kordower, 1999). In a recent study, OEC transplants did not improve hindlimb locomotion following a contusive spinal injury in rats, although SC transplants were effective (Takami et al., 2002). Therefore, the ideal treatment for individual spinal cord patients may depend on the nature of their injury. Future studies should investigate the efficacy of OEC transplants across injury types, age of injury, etc. In some cases, OECs may be most effective when used in combination with other treatments. Transplants of SC "bridges" across spinal
lesions have been very successful when OECs were co-transplanted at the ends of the bridge (Ramon-Cueto et al., 1998). This technique takes advantage of the therapeutic benefits of both cells. Here, OECs play the important role of guiding axonal growth between the graft and the CNS (see Figure 3). OEC and SC transplants need to be tested in conjunction with other therapies, such as IN-1 antibodies (Schnell and Schwab, 1990), to discover which combinations are most beneficial.
Despite the many questions about OECs that remain unanswered, some researchers are anxious to move into clinical trials of OEC transplants. In fact, a group of Australian scientists began Phase I clinical trials in June, 2002 (Senior, 2002). In this study, OECs were removed from the nasal tissue, purified, cultivated, and autotransplanted into the spines of four paraplegic patients. The purpose of the trial is to assess the safety of OEC transplants, but functional changes will be monitored over the next three years. Some scientists agree with the decision to begin OEC transplants in humans, given the success of OEC transplants in rodents. Others believe that this controversial clinical trial is too early. They argue that transplants of human OECs should be tested in other animals to determine the generalizability of rodent results. Smith and colleagues have suggested that we use dogs as an intermediate model because their spinal cords are similar in size to humans, and accidental spinal cord injuries are
common among domestic dogs. They have shown that transplantation of OECs from canine olfactory bulbs can remyelinate the spinal cord in rats (Smith et al., 2002), but the effects on SCI are not yet tested. Other groups are developing monkey models of SCI (Perry et al., 2002).
The excitement over OEC transplants is understandable. But without the proper background research, clinical trials may disrupt natural recovery, cause dangerous tumors, or simply be ineffective. Clinical "null results" are often accompanied by increased opposition from Ethics committees, a damping of scientific interest, and the possibility of dangerous side-effects. Therefore, we should be very confident in our understanding of OECs before transplanting them within humans. In the meantime, we must exercise patience as we continue with preclinical trials and in vitro research. This will increase our chances of reaching a cure for SCI in the long run.