Myelin damage and repair in pathologic CNS: challenges and prospects

1Myelin is a cholesterol rich extension of oligodendrocytes and Schwann cells (SCs) plasma membrane, which serves as a specialized insulation sheath for axons in the nervous system. Myelin facilitates axon signal conduction through enabling “saltatory conduction” (see review by ). However, the importance of myelin in the central nervous system (CNS) is beyond its role in rapid signal conduction along axons as its disturbance also cause other severe functional and neurobehavioral disabilities (as reviewed by ). Myelin is important for axon maintenance and function. Perturbations of myelin structure and function or “demyelination” is associated with a long list of CNS pathologies from congenital and autoimmune disorders to metabolic disturbances. Progressive demyelination also results in axonal degeneration due to the disruption of axo-oligodendrocyte signaling. A healthy cross talk between axons and oligodendrocytes is required to maintain proper metabolic function of axons, trophic support, cytoskeletal arrangement, ion channel organization, and axonal transport. Axons become dependent on myelinating glia as myelin appears during the development. This concept was demonstrated in PLP/DM20 deficient mice where the absence of these essential myelin proteolipids resulted in axonal swellings only in myelinated axons with no evidence of axonal pathology in normal unmyelinated fibers. Moreover; mice lacking Cnp1, which encodes 2′,3′-cyclic nucleotide phosphodiesterase in oligodendrocytes, show no structural abnormality in myelin but develop axonal swelling and degeneration. These studies suggest that myelinated axons receive signals from oligodendrocytes that trigger their dependency to myelin. Interestingly, such a dependency have not yet been observe in non-myelinated axons. Survival of oligodendrocytes is also dependent on axons. Following injury, oligodendrocytes distal to the site of an axonal injury degenerate due to lack of trophic support from the injured axon. Considering the reciprocal axo-oligodendrocytes signaling, replacement of oligodendrocytes and renewal of myelin sheath around surviving demyelinated axons following injury is a vital repair strategy for CNS regeneration and functional recovery.

2Oligodendrocyte precursor cells (OPCs) and neural stem/progenitor cells (NPCs) are two endogenous cell populations, capable of replacing lost oligodendrocytes and remyelinating spared axons following injury. Despite the spontaneous response and activation of both OPCs and NPCs upon injury, adequacy, and quality of remyelination is challenged due to multiple factors including modifications in the extracellular matrix, astrogliosis, and downregulation of essential trophic and growth factors. These injury-induced events either limit oligodendrocyte differentiation or impede the process of axonal ensheathment and remyelination. Over the last decade, cellular and pharmacological repair strategies have been developed to induce remyelination by recruiting endogenous precursor cells or through stem cell therapies. In this review, (1) we will provide an overview on the precise molecular and ion channel organization of myelinated axons and the reciprocal axo-myelin interactions that warrant properly balanced physiological activities within the CNS, (2) we will dissect the underlying cause of demyelination and the structural and functional consequences of demyelination in axons by focusing on spinal cord injury (SCI) and multiple sclerosis (MS) models, (3) we will discuss the role of activated glia in demyelination and remyelination following demyelination, and (4) we will review the current status of cell-based therapeutic interventions that are designed to promote oligodendrocyte differentiation and facilitate remyelination. Understanding the functional ramification of demyelination and remyelination and the cellular and molecular basis of these events will aid in developing targeted therapies to more effectively promote myelin repair and prevent disease progression in demyelinating conditions.

3Myelin is a modified plasma membrane of oligodendrocytes in the CNS, which enwraps a segment of axon in a spiral fashion. Myelination affects function and molecular organization of axons allowing faster signal propagation with reduced energy consumption. Several proteins in myelin have been identified to play essential roles in axonal maintenance and function. Proteolipid protein (PLP) and its spliced derivative, DM20, are essential for proper axonal function. Loss of either PLP or DM20 will affect myelin periodicity and cause axonal swelling. Swollen axons will gradually become dysfunctional and degenerate causing functional deficits at later stages. Myelin basic protein (MBP) is another structural protein that plays a vital role in myelin compaction and thickening in the CNS. Shiverer mice that lack MBP demonstrate dysmyelinated axons associated with axonal dysfunction and motor impairments. Interestingly, Shiverer mice do not develop axonal swelling and show minimal axonal degeneration compared to PLP/DM20 deficient mice even up to 2–3 months following birth. Myelin associated glycoprotein (MAG) is essential for the initiation of myelination. Mice with double knockout of MAG and Fyn (a downstream signaling molecule in MAG/Fyn pathway) demonstrate severe optic nerve hypomyelination despite the unaffected presence of oligodendrocytes. MAG is also known to be essential for survival and integrity of myelinated axons, however, such a role has not been established for Fyn. CNPase (2,3-cyclic nucleotide 3-phosphodiesterase) is an enzyme that is synthesized in myelinating mature oligodendrocytes and can be found in non-compact regions of the myelin sheath. Lack of CNPase has not been shown to affect myelination but myelinated axons will eventually become swollen and degenerate. This evidence demonstrates the importance of the various myelin compartments/proteins for the proper functioning of axons and oligodendrocytes. However, further investigations are required to elucidate the role of each myelin protein in this complex relationship.

4Myelinated axons show a high degree of structural organization. A myelinated axon can be separated into distinct domains including node of Ranvier, paranode, juxtaparanode, and internode (Figure 1A). Node of Ranvier is the gap between two adjacent myelin sheaths and contains high concentrations of voltage-dependent Na+ channels on the axonal membrane. Electrical impulse cannot flow through the high resistance myelin sheath, but instead flows through the node of Ranvier and depolarizes the axonal membrane at each node resulting in saltatory conduction.

5In myelinated axons, node of Ranvier was characterized by the localization of voltage-gated sodium (Nav) and KCNQ K+ channels. Node of Ranvier also contains a collection of adhesion molecules, adaptor proteins, and cytoskeletal structures including, βIV-spectrin, ankyrin G, neuron-glia-related cell adhesion molecule (NrCAM) and a 186 kDa isoform of neurofascin (NF186) (Figure 1A). Among these molecules, βIV-spectrin and ankyrin G play a major role in stabilizing the Nav channels at nodal region. During the development of axons, Nav1.2 channels are initially expressed along pre-myelinated axons with the capability to generate an action potential. As myelination ensues, Nav 1.6 channels begin to cluster at mature nodes of Ranvier. Nav1.2 and Nav1.6 channels are both rapidly activating and inactivating channels but Nav1.6 is known to produce a larger persistent current. Glial cells play an essential role in the formation of normal nodes of Ranvier with their typical nodal Nav and paranodal Kv channel distribution. As it has been reviewed by, there are similarities in the contribution of glial cells in node formation between the CNS and PNS. In both systems glial cell adhesion molecules (CAMs) in close association with axonal CAMs and cytoskeletal domain, form a structural framework that clusters ion channels with specific formation in nodal and paranodal areas (see Figure 1A).

6Paranode is the adjacent segment to the node of Ranvier where myelin loops provide an anchor by tethering the myelin to the axonal membrane. Evidence has established a determining role for paranodal axo-oligodendrocyte junction in precise localization of ion channels into specialized domains of myelinated axons. Paranodal junctions are critical in preventing lateral diffusion of ion channels along the axons to ensure proper segregation of Na+ and K+ channels at discrete domains on axonal membrane. The paranodal region was characterized by the presence of Contactin and Contactin-Associated Protein (Caspr) that form a complex in the “septate-like junctions” between myelin loops and axolemma. Caspr is critical for the establishment of axo-glia junction in myelinated fibers through its interactions with contactin and NF-155. Caspr deficiency results in disruption of the paranodal region and aberrant distribution of ion channels along the axons (see Figure 1A).

7The juxtaparanode contains delayed-rectifier voltage-gated Kv+ channels and Na+/K+ ATPase channels that allow for rapid exchange of axoplasmic Na+ for extracellular K+ and restoration of the resting membrane potential. As ion channel clustering evolves, shaker type Kv1.1 and Kv1.2 channels begin to localize in juxtaparanodal region of the myelinated axons. These channels are associated with the Caspr2/TAG-1 adhesion complex (Figure 1A). Upon receiving of an action potential, Nav channels open, allowing an influx of Na+ into the axon causing depolarization. After each depolarization, Na+/K+ ATPase pumps, located at the juxtaparanodal and internodal regions, exchange axonal Na+ for extracellular K+. This process is an energy dependent mechanism and is essential for rapid and repetitive axonal firing. Similarities exist between the molecular organization of nodes and the axon initial segment; however, while myelin is crucial for the proper molecular organization of nodes, the axon initial segment appears to be intrinsically organized by the neuron. Evidence from our group and others have shown that demyelination due to injury and disease results in disruption of the precise nodal organization causing axonal dysfunction. Additionally, myelination provides extrinsic trophic signals, which influence the normal maturation, maintenance, and long-term survival of axons. Structural and functional importance of nodal organization will be discussed in subsequent sections.

8Demyelination is damage or loss of the myelin sheath around axons. It is mainly a consequence of oligodendroglia cell death that can occur through multiple mechanisms depending on the type of disease or injury, including genetic defects, infectious agents, autoimmune reactions, trauma, and some by unknown mechanisms. Several genetic disorders exist that can cause defects in myelin through improper myelination and myelin maintenance or progressive demyelination over time. Charcot-Marie-Tooth disease (CMT), Alexander disease, and Krabbe disease are examples of the many known genetic diseases characterized by axonal demyelination or dysmyelination.

9Multiple Sclerosis is a classic example of autoimmune demyelination in the CNS. The early stages of MS involve relapsing-remitting where patient experience demyelination associated with loss of function (i.e., vision and gait), which is usually regained following remyelination. In the progressive stages of MS, irreversible functional deficit occurs which has been associated with progressive loss of axons and neurons. Degeneration of chronically demyelinated axons is now considered to be a major contributor to the permanent neurological disability that MS patients eventually endure.

10Demyelination can also occur through traumatic injury. In the chronically injured spinal cord, there is varying degree of demyelination and dysmyelination in the subpial rim surrounding the lesion site. Following SCI, some axons and oligodendrocytes are initially lost through necrosis due to mechanical injury. As injury evolves, progressive loss of oligodendrocytes occurs through apoptosis and autophagy that results in demyelination of injured spared axons. Remyelination occurs spontaneously by both OPCs and NPCs following injury even in the chronically injured spinal cord. However, this remyelination attempt is often limited and inadequate due to changes to the post-injury environment. Therapeutic strategies aimed at promoting remyelination have demonstrated the potential to promote axonal sparing and limit progressive axonal dieback in chronic SCI.

11Animal models of demyelinating disease such as MS provide invaluable tools to study myelin–axon interactions and understand the pathological effects of demyelination on axonal integrity and function. Here, we will primarily focus on the effects of demyelination on axons in models of MS and SCI, however, many of the details provided here also correlate with other findings in the literature in other demyelinating conditions.

12Loss of myelin sheath causes aberrant distribution of ion channels, where Nav channels diffuse away from the nodes and redistribute across the axonal surface. Additionally, there appears to be an overall increase in the expression of Nav channels in chronically demyelinated axons. Following experimental autoimmune encephalomyelitis (EAE), Nav1.2, and Nav1.6 channels are up-regulated in demyelinated axons (see Figure 1B). Sodium channel redistribution causes an overall increase in Na+ influx during impulse conduction and increased demand for ATP during repolarization. Furthermore, increased sodium influx has been associated with axonal degeneration through a Ca2+-mediated effect by causing the reversal of the Na+/Ca2+ exchanger (NCX) (; Figure 1). Inhibition of sodium channels and NCX has been shown to prevent axonal degeneration. Increased axonal Ca2+ can activate proteolytic enzymes and eventually lead to degeneration of chronically demyelinated axons. Ca2+ influx is not normally toxic in axons, however, increased energy demand and thus lack of ATP in axons following demyelination causes failure in the energy-dependent Ca2+ buffering system to efficiently remove excess Ca2+. This results in a rise in the Ca2+ concentration to toxic levels. Lack of ATP in chronically demyelinated axons is thought to render axons vulnerable to cellular death over time.

13Axonal degeneration caused by sodium influx is thought to be mainly mediated through Nav1.6 channels. Nav1.6 channels produce a persistent current, which is much larger than that of Nav1.2. Nav1.6 has been shown to be co-localized with NCX following demyelination of axons in EAE model and in postmortem cervical spinal cord and optic nerve tissue of acute MS patients. Nav1.6 is co-localized in 60% of axons, which express β-amyloid precursor protein (APP), a marker for axonal injury, whereas it is only expressed in 20% of axons, which are β-APP negative. Importantly, in chronic lesions of MS, Nav1.6 is expressed in patches in only a third of axons. Evidence shows that demyelinated axons are more susceptible to axonal injury than dysmyelinated axons, which may be explained by the altered expression of Nav1.6. Dysmyelination in MBP-/- Shiverer mice have altered expression of Nav channels in their axons with Nav1.2 being retained into adulthood and Nav1.6 not being expressed. Similarly, PLP-/- mice also show a loss of Nav1.6 channel clustering and an increased expression of Nav1.2. Because Nav1.2 channels are expressed in pre-myelinated axons and can produce signals, it is believed that newly produced Nav1.2 channels support conduction in demyelinated axons.

14Juxtaparanodal voltage-gated Kv+ channels are also influenced by demyelination after SCI or genetic dysmyelination. We have shown that spinal cord axons in dysmyelinated Shiverer mice exhibit a dispersed distribution of Kv+ channel subunits Kv1.1 and Kv1.2 associated with the loss of the characteristics of juxtaparanodal and paranodal structures (Figure 1B). Our investigations on dysmyelinated Shiverer mice and Long Evans Shaker (LES) rats elucidated the role of K+ channels in axonal function. These studies revealed aberrant localization and increased expression of both Kv1.1 and Kv1.2 channel subunits along axolemma of dysmyelinated axons while these channels were confined to juxtaparanodal regions of the wild-type axons. Using sucrose gap recording of spinal cord monophasic compound action potentials (CAP), we demonstrated that Shiverer spinal cord axons have significantly lower CAP amplitude and area compared to wild-type counterparts. Kv+ channel blockage by specific (DTX-I, DTX-K) and non-specific (4-AP) blockers improved axonal conductance; however, this effect was shown to be more dependent on a combination of subunits as a specific blocker of Kv1.1 failed to improve axonal conduction significantly. Interestingly, Kv+ channels are also important for remyelination. Administration 4-aminopyridine (4-AP), a broad-spectrum K+ channel antagonist that blocks fast K+ channels, to a mouse model of cuprizone-induced demyelination, resulted in impaired oligodendrocyte regeneration and remyelination. These studies collectively demonstrate the pivotal role of axo-myelin interactions in ion channels distributions and functions and more importantly on axonal physiology.

15Axonal transport shuttles critical cell body-derived components back and forth between the soma and axon and across synapses in neurons. Dysfunction of axonal transport causes neuronal homeostasis imbalance and as a result makes axons more susceptible to axonal degeneration. Axonal transport disturbances are thought to precede the initiation of neurodegeneration in diseases including, hereditary spastic paraplegias (HSPs), AD and Huntington’s disease. Accumulation of APP is known as; an early marker of injury in MS patients and is believed to accumulate due to lack of axonal transport following injury.

16Oligodendrocytes and their myelin sheath are critical in regulating slow and fast anterograde transport rates. Reduced fast axonal transport can cause degeneration in distal parts of the axons as observed in X-linked spastic paraplegia type 2, which is caused by a mutation of the PLP1 gene, a major protein of the myelin sheath. Absence of PLP causes swelling of axons and deficits in retrograde and anterograde transport. In both humans and mice, absence of PLP causes selective axonal degeneration of long tracts including the fasciculus gracilis and distal corticospinal tracts. Conversely, Shiverer mice, lacking MBP, demonstrate a significant increase in slow axonal transport associated with increased density and instability of microtubules in axons.

17Demyelination-induced defects in axonal transport has been also detected in MS models. Studies in an EAE model of optic neuritis in rats suggest that the extent of disruption in axonal transport appear to be correlated with the severity of inflammation, demyelination, and axonal injury. Live imaging of individual axonal organelles in the spinal cord of mice with acute EAE revealed that the anterograde and retrograde transport of mitochondria and peroxisomes were markedly reduced in spinal axons, which passed through the lesion. In chronic model of MS, when axonal transport is restricted, there is reduced density in distal organelles which can ultimately lead to “starvation” of distal axonal arbors and axonal degeneration. Transport deficits were shown to occur prior to any marked alteration of microtubule tracks. Dysfunction of axonal transport recovers within days following insult. However, in chronic MS lesions, transport deficits were apparent resulting in lack of distal organelle supply. Reduced axonal transport has been attributed to reactive oxygen and nitrogen species released by immune cells, which alters the attachment of motor/cargo complexes to microtubules. In chronic MS models, anterograde transport from the soma to the synapses appears to be considerably affected resulting in reduced organelle transport from cell body to the axonal terminal at synapses. Hence, interventions to restore transport tracks and axonal transport seem to be vital strategies to slow down the progression of axonal degeneration and reverse these degenerative effects.

18The majority of white matter tract axons were entirely enwrapped by myelin; therefore, it is likely that axons cannot obtain proper nutrient support from their external environment on their own and require metabolic support from glial cells. It is currently unknown whether glucose transporters are present at the node of Ranvier. In axons, the highest energy requirement occurs at the Na+/K+ ATPase pump, which is located along the internodes. It is proposed that axons receive their metabolic support mainly through the “pan-glial” network of oligodendrocytes and astrocytes. Astrocytes were further connected to the blood–brain-barrier and to the nutritive support by brain capillaries. Oligodendrocytes, expressing connexin (Cx) 47, were coupled to astrocytes, expressing Cx30, through gap-junctions. Double KO mice of Cx47 and Cx30 results in axonal loss, and death in mice. This evidence suggests that myelination has a dual role in supporting the metabolic activity of neurons by saving energy of axons through saltatory conduction and providing nutrients to neurons.

19Ensheathment of axons by oligodendrocytes are shown to drastically diminish the ATP consumption of neurons by reducing the energy required by axons to transmit signals over long distances through saltatory conduction. However, is it important to take into consideration that the metabolic cost of myelin synthesis and maintenance might be higher than the saved energy. Nonetheless, myelin does save the amount of energy required in neurons by decreasing the energy expenditure required to maintain its resting membrane potential and to propagate signals. Following demyelination, there is an overall increased demand for ATP. The energy required to maintain the intra-axonal ionic balance and the resting membrane potential by the Na+/K+ ATPase pump were increased, due to redistribution and an overall increase in the number of Na+ channels. Shiverer mice exhibit a significant change in the density and activity of mitochondria in their axons in comparison to wild type animals. Similarly, unmyelinated segments of retinal ganglion cell axons in the lamina cribrosa also show increased metabolic activity in comparison to myelinated segments. Increased metabolism requirements for demyelinated axons may make these axons more susceptible to death through disease mechanisms such as inflammation.

20Mitochondria are the major source of axonal ATP and play a critical role in apoptosis, reactive oxygen species generation and calcium buffering. Two separate populations of mitochondria exist in myelinated axons, stationary and motile mitochondria. The majority of mitochondria are located throughout the axons in stationary sites where multiple mitochondria reside. Stationary mitochondrial are predominantly located in juxtaparanodal/internodal axoplasm, containing multiple morphologies of varying length, whereas those located in the nodes/paranodal axoplasm appear uniformly short or absent altogether. Energy consumption in axons is highest at the juxtaparanodal and internodal regions where Na+/K+ ATPases are abundantly present. These stationary mitochondria do not translocate and usually vary in length but typically contain the same diameter throughout the population. A separate population of relatively small but motile mitochondria also exist which translocate in both anterograde and retrograde directions. These motile mitochondria are produced in the cell body, and can stop within stationary sites. They are essential for the turnover and redistribution of mitochondria and have been shown to fuse with or bud from stationary mitochondria. The rate of transport and docking of these motile mitochondria can be influenced by axonal metabolic demand, such as increases in axonal firing.

21Recent evidence has shed some light onto the changes that occur to mitochondria in the acute stages of demyelination. Following demyelination there is an overall increased demand for ATP mainly due to changes in ionic homeostasis. Moreover, the size of stationary sites and the speed of mitochondrial transport is increased in demyelinated axons. In vitro studies on myelinated rat dorsal root ganglion (DRG) axons showed 2.2-fold increase in the size of stationary mitochondria sites and 47% increase in the velocity of motile mitochondria following demyelination. This response is shown to be an axonal response to the increased ATP demand of demyelinated axons mediated, at least partially, through activating transcription factor 3. Increased volume of mitochondria at these stationary sites are shown to be a protective response by demyelinated axons mediated through syntaphilin, a protein, which tethers mitochondria to microtubules at stationary sites. Chronically demyelinated axons exhibit increased expression of syntaphilin. Demyelinated axons deficient in syntaphilin degenerate at a significantly greater rate than wild type axons associated with smaller increases in stationary mitochondrial volume indicating the importance of mitochondrial migration to these stationary sites. In summary, increasing mitochondrial stationary site size is important in protecting neurons from degeneration following CNS demyelination.

22Despite this protective response from axons, mitochondrial function appears to be limited in chronically demyelinated lesions of MS. Following demyelination, changes to the energy balance in axons and dysfunctions of axonal mitochondria contribute to degeneration of chronically demyelinated axons. There appears to be an overall decrease in the ability of neurons to produce ATP through their mitochondria. In postmortem MS tissues, there was a decreased expression of mitochondrial electron transport chain genes which was associated with decreased ability of mitochondria to exchange electrons in respiratory chain complex I, III, and IV. This decrease in respiration was later shown to be mediated through deletion of mitochondrial DNA in axons. These data suggest that mitochondria in the chronically demyelinated axons have a reduced ability to produce ATP, which can contribute to the axonal degeneration over time.

23It is well-known that spontaneous remyelination occurs naturally after CNS injury. However, the extent and quality of remyelination is limited following injury resulting in limited reorganization of nodes of Ranvier and continued axonal dysfunction. In rat compressive chronic SCI, we found considerable number of chronically injured axons in the rim of white matter that exhibited aberrant distribution of Kv1.1, Kv1.2, and Caspr along the paranodal and juxtaparanodal regions, an established characteristic of axonal demyelination or dysmyelination. Additionally, electron micrographs of the injured white matter showed that the spontaneous remyelination after SCI is suboptimal and incomplete as the newly formed myelin around the injured axons is thinner than normally myelinated axons. Considerable evidence over the past years has uncovered that failure of the injured and diseased spinal cord for adequate remyelination is attributed to multiple factors that include (1) the limited replacement of myelinating oligodendrocytes by spinal cord progenitor cells, (2) insufficient levels of key growth factors for oligodendrocyte maturation and myelination, (3) inadequate clearance of myelin debris that interferes with the process of axonal ensheathment and remyelination, (4) inhibitory factors mainly driven by activated glia that inhibit migration and maturation of OPCs, differentiation of NPCs to oligodendrocytes, and axonal ensheathment. In the following sections, we will discuss endogenous mechanisms of remyelination and the role of injury microenvironment in modulating the replacement of oligodendrocytes and axonal remyelination.

24Spinal cord injury results in loss of oligodendrocyte population acutely due to necrosis caused by the primary tissue damage. However, oligodendrocyte cell loss continues progressively through apoptosis-mediated cell death at subacute and chronic stages of SCI. Evidence shows that oligodendrocytes are highly susceptible to cell death even after moderate contusive injury resulting in the loss of over 90% of the oligodendrocytes at the lesion epicenter by seven days after injury. Interestingly, apoptotic oligodendrocyte death is also observed chronically along the long fiber tracts as a consequence of axonal degeneration and loss of trophic support from axons. Similar process has been observed chronically in primate models of contusive SCI and in human SCI. Multiple secondary injury mechanisms contribute to oligodendrocyte loss in SCI including digestion by proteolytic enzymes released from damaged cells and toxic blood components, ischemic damage, and oxidative stress, glutamate and ATP mediated excitotoxicity, pro-inflammatory cytokines released from infiltrated neutrophils and lymphocytes and autophagy.

25Despite extensive cell death, new oligodendrocytes form and remyelination occurs spontaneously following SCI and demyelinating CNS diseases. Mature oligodendrocytes are post-mitotic and unable to contribute to cell renewal. However, the spinal cord harbors a population of adult OPCs that contribute to oligodendrocyte replacement following injury. These OPCs can be identified by the expression of platelet derived growth factor receptor α (PDGFR-α) and NG2 proteoglycan. Recent findings have shown that resident adult spinal cord OPCs become activated, and change their gene transcription pattern resembling immature OPCs. OPCs differentiate into myelinating oligodendrocytes and remyelinate spared axons following demyelination. In addition to OPCs, the spinal cord also contains a population of endogenous NPCs, which is known to contribute to oligodendrocyte replacement following injury. These NPCs exist in the ependymal layer of the intact spinal cord. In adulthood and under normal conditions, NPCs are latent and their activity is mainly to maintain their own population through self-renewal. However, upon injury, they become activated and migrate to the site of injury where they can generate new glial cells. Studies by our group and others have demonstrated that activated NPCs predominantly differentiate into astrocytes after SCI, with limited number differentiating into new oligodendrocytes. Moreover, the newly formed NPCs derived astrocytes contribute to glial scar formation following SCI.

26Recent studies have shown that limited ability of NPCs for oligodendrocyte differentiation in SCI milieu may be attributed to unavailability, or modified expression of essential growth factors for oligodendrocyte development. Several studies have addressed this possibility by administering growth factors to optimize the post-SCI microenvironment to support survival and differentiation of transplanted and endogenous NPCs into oligodendrocytes as well as remyelination. In our NPC transplantation studies in subacute and chronic SCI, sustained infusion of a cocktail of growth factors including EGF, bFGF, and PDGF-AA was able to significantly enhance the long-term survival of NPCs in the injured spinal cord. Importantly, transplanted NPCs were able to survive and integrate within the host tissue and differentiate into mature myelinating oligodendrocytes and remyelinated axons. EGF and bFGF are known to play essential role in NPC survival and proliferation. In vivo delivery of these growth factors into animal models of contusive SCI was associated with increased proliferation in ependymal layer where NPCs reside. However, despite increased proliferative activity, no significant change in oligodendrogenesis were seen, which could be due to the lack of PDGF-AA in this growth factor regimen. PDGF-AA promotes the proliferation of glial progenitor cells and can trigger differentiation and survival of newly formed oligodendrocytes. Moreover, bFGF in synergy with PDGF can regulate proliferation of adult OPCs. Interestingly, a developmental study on FGF receptor 1 and 2 double knockout mice (Fgfr1-/-, Fgfr2-/-) showed normal OPC proliferation, differentiation and initiation of myelination. However, these FGF receptor null animals demonstrated defective myelin thickening during postnatal period and remained defective throughout their adulthood. This evidence suggests that FGF signaling can regulate myelin sheath thickness.

27Mash1, a transcription factor known to promote neural differentiation, have also been implicated in endogenous oligodendrocyte differentiation. Retroviral induction of Mash1 expression in endogenous spinal cord NPCs following SCI resulted in increased oligodendrocyte differentiation and formation of new oligodendrocyte progenitor cells following a complete transection rat model of SCI. Although a small number of new Mash1 expressing oligodendrocytes expressed markers of mature oligodendrocytes, the majority of these cells remained NG2+ expressing progenitor cells and did not fully mature into myelinating oligodendrocytes even weeks after injury.

28Neuregulin-1 (Nrg-1) is another growth factor known to promote OPCs survival, migration, and differentiation into mature myelinating oligodendrocytes. Nrg-1 is known to play essential roles in oligodendrocyte and SC myelination. Our group has recently demonstrated that the rapid and long lasting downregulation of Nrg-1 following contusive SCI is an underlying cause of inadequate oligodendrocyte differentiation. Restoring the reduced levels of Nrg-1 in the injured spinal cord enhanced tissue preservation, oligodendrocyte differentiation of spinal cord NPCs, and increased oligodendrocyte and axonal survival following SCI. Collectively, these studies show the necessity of micro-environmental optimizations in order to improve endogenous and exogenous replacement of oligodendrocytes and axon remyelination following SCI.

29Current evidence shows that remyelination is additionally limited by inhibitory modifications in the post-SCI niche caused by secondary injury mechanisms particularly in chronic SCI. Newly formed oligodendrocytes often fail to fully ensheath and myelinate the injured spared axons following injury resulting in incomplete remyelination. These inhibitory signals are primarily associated with myelin debris, activated glial cells, and infiltrating leukocytes following injury.

30Presence of myelin debris and insufficient clearance by microglia and macrophages contributes to incomplete remyelination by inhibiting OPCs differentiation and maturation in vitro and in vivo. Recent in vitro studies by indicate that exposure to myelin debris prevents OPCs maturation and their transition to a myelinating phenotype. This was demonstrated by a significant decrease in the number of mature oligodendrocytes and was accompanied by increased expression of two proteins, namely inhibitor of differentiation (ID) 2 and ID4 that are known to block oligodendrocyte maturation. It has been shown that myelin clearance and remyelination become less sufficient with aging due to changes in macrophage secretory and phagocytic activity. A study using a technique known as “heterochronic parabiosis” where the circulation of a young animal is infused into an older animal, demonstrated improvements in remyelination in the old animal which is presumably due to better functioning of young circulating monocytes for myelin debris clearance. Myelin debris is a potent inhibitory component of injured spinal cord that impairs regeneration and remyelination. Thus, proper myelin clearance is an important step for remyelination process.

31Other molecules and pathways known to inhibit myelination include LINGO (leucine rich repeat and Ig domain-containing, Nogo receptor interacting protein), Wnt signaling, and Semaphorin 3A (Sema3A). LINGO-1 is a component of Nogo receptor signaling complex. In a hemisection model of SCI, application of LINGO-1 antagonist (LINGO-1-Fc) promoted functional recovery. Dysregulation of Wnt signaling in OPCs also inhibits myelination during development and repair. Wnt signaling is activated in differentiating OPCs following chemically induced demyelination and in samples from MS patients. Following demyelination, upregulation of T-cell factor 4 (tcf4), a Wnt pathway mediator, is significantly upregulated in differentiating OPCs and inhibit oligodendrocyte maturation and myelination. Similarly, Sema3A negatively affects OPC maturation and recruitment in demyelinating conditions. Its level increases significantly after SCI, reaching its peak at one week following injury. Increased expression of Sema3A has also been observed in MS and experimental demyelination models. Sema3A overexpression delays recruitment of OPCs to the demyelination site through a chemo-repulsive mechanism. Use of Sema3A inhibitor improved tissue preservation, remyelination and functional recovery following SCI.

32Collectively, these findings demonstrate that endogenous remyelination was impeded by the inhibitory microenvironment following injury and activated astrocytes and microglia/macrophages seem to play pivotal roles in this inhibition. We will discuss recent studies on the role of resident glial cells and peripherally recruited immune cells in modulating oligodendrocyte replacement and remyelination following CNS injury.

33Astrocytes play critical role in several aspects of myelination in pathologic CNS including clearance of myelin debris, modulating the activity of oligodendrocytes, myelin maintenance, and renewal. Using a cuprizone model of rodent demyelination, demonstrated that astrocytes contribute to the clearance of myelin debris by inducing the recruitment of microglia into demyelinated lesion sites. Astrocytes impose their modulatory effects through upregulation of CXCL10, a chemokine that is known to play a role in T-cell chemoattraction in CNS autoimmune disorders such as MS.

34Intercellular connections between astrocytes and oligodendrocytes are critical for the proper physiology of oligodendrocytes. While there are no gap junctions between oligodendrocytes themselves, they are connected to astrocytes through gap junctions, which make oligodendrocytes indirectly interconnected. Evidence shows that gap junctions are essential for proper myelin physiology in the CNS. Four different types of connexins have been identified in oligodendrocytes (Cx29, 32, 45, 47). Cx29, Cx32, and Cx47 are known to be expressed by oligodendrocytes that in conjunction with Cx26, 30, 43 on astrocytes, form the astrocyte-oligodendrocyte gap junction complex. Double knockout mice models lacking Cx47 and Cx32 die postnatally due to severe apoptotic oligodendrocyte death, hypomyelination, and axonal degeneration. This evidence suggests a critical role for astrocyte and oligodendrocytes inter-cellular signaling in myelin physiology.

35Astrocytes provide trophic support to oligodendrocytes by producing growth factors. In an ethidium bromide (EB) induced rat model of spinal cord demyelination, were able to show despite recruitment of OPCs to the site of injury, they failed to mature and remyelinate axons in the areas that astrocytes were absent. Astrocytes are known to produce PDGF and LIF, which are supportive for oligodendrocyte survival at progenitor and mature stages, respectively.

36While supportive of myelination in the normal CNS, astrocytes can play detrimental roles in CNS remyelination following pathology. Astrocytes contribute substantially to the extracellular matrix of the CNS. Following injury, they are activated and form a glial scar, which is inhibitory to the repair and regeneration of the CNS. The inhibitory influence of scar is mediated mainly through chondroitin sulfate proteoglycans (CSPGs), which have known inhibitory effects on axonal regeneration, axonal conduction, remyelination, and cellular therapies in SCI (;,;;;;; for review see ). Our recent evidence shows that CSPGs inhibit the ability of NPCs to proliferate, spread their cell processes, survive and differentiate into oligodendrocytes. Degradation of CSPGs with chondroitinase ABC promotes oligodendrocyte differentiation and myelination of both transplanted NPCs and endogenous precursor cell populations. The detrimental effect of CSPGs upregulation by astrocytes is also observed in MS lesion where the CSPGs aggrecan, neurocan, and versican as well as hyaluronan accumulate at the borders of active demyelinating lesions. In vivo and in vitro observations have shown that CSPGs limit the ability of OPCs to migrate, mature and myelinate axons. Removal of CSPGs is correlated with enhanced remyelination in MS lesions. Collectively, these data identify the inhibitory role of activated astrocytes and scar-associated CSPGs, in modulating NPCs and OPCs integration, migration, maturation and myelination in SCI and MS conditions. In addition to the inhibitory ECM produced by astrocytes, reactive astrocytes can also be detrimental to remyelination in demyelinated CNS through the secretion of Endothelin-1. Endothelin-1 is shown to inhibit the differentiation of OPCs into mature myelinating oligodendrocytes through the activation of Notch signaling.

37Taken together, these data demonstrate the complex role of astrocytes in the CNS. The presence of astrocytes is required to produce healthy myelin, however, the detrimental effects of activated astrocytes and their production of inhibitory ECM molecules following injury limits the ability of the CNS in self-repair and axon remyelination. Thus, developing interventions to moderate the inhibitory effects of scar-associated molecules is a vital therapeutic strategy for CNS repair and remyelination following injury.

38Emerging evidence indicates that macrophages and microglia also play critical roles in modulating demyelination and remyelination through their antigen presenting ability and production of cytokines, chemokines and growth factors (for review see;; ). After CNS injury or infection, microglia/macrophages undergo phonotypical changes and become polarized into pro-inflammatory “classically activated” M1 or anti-inflammatory “alternatively activated” M2 phenotypes (as reviewed by;; ). Although both M1 and M2 cell types are activated microglia/macrophages, they play distinct roles in CNS injury and repair. Pro-inflammatory M1 microglia/microphages are characterized by the production of cytokines such as interleukin (IL)- 1β, IL-6, IL-12, tumor necrosis factor (TNF)-α and reactive oxygen and nitrogen species such as nitric oxide (NO). Conversely, M2 microglia/microphages are a source of anti-inflammatory factors such as arginase-1 (Arg-1) and IL-10, which are known for their role in the development of type II adaptive immune responses. Generally, accumulating evidence has identified a pro-regeneration role for M2 microglia/macrophages including a supporting role in overcoming axonal growth inhibition imposed by CSPGs and myelin debris.

39A recent study also demonstrated phonotypical changes in macrophages/microglia following lysolecithin-induced demyelination in mice. Using specific M1/M2 markers, identified a “switch” from M1 to M2 phenotype following demyelination. This transformation occurred between day 3 to 10 post-lysolecithin-induced demyelination when M1 dominant population of CD68+ macrophages/microglia adopted a M2 dominant phenotype identified by Arg-1 expression. This time window was closely correlated with a regenerative stage at which OPCs were recruited to the site of lesion, and differentiated into mature myelinating oligodendrocytes. Further in vitro and in vivo investigations confirmed a supportive role for M2 microglia/macrophages in remyelination. Adding M2 conditioned media into OPCs cultures increased oligodendrocyte differentiation and maturation. Selective depletion of M1 macrophages by intralesional injection of gadolinium chloride reduced the proliferation rate of OPCs without affecting their migration and remyelination capacity. Interestingly, in a rat model of lysolecithin demyelination, M2 depletion was associated with delayed oligodendrocyte differentiation and nodal reconstruction suggesting the pro-myelinating role of M2 macrophages.

40Activation of microglia through intraspinal injection of lipopolysaccharide (LPS), a Toll-like receptor-4 (TLR-4) agonist, caused a significant increase in NG2+ cell proliferation and oligodendrocyte differentiation. However, activating microglia using intraspinal injection of zymosan, a TLR-2 agonist, showed oligodendrocyte loss without increase in NG2+ cell proliferation. Interestingly, the extent of SC remyelination remained unaffected. Of note, these studies also revealed that the timing of macrophages response is a key factor as the early presence of the macrophages was important for remyelination while delayed macrophage depletion did not impair remyelination.

41Several mechanisms have been proposed for the positive role of macrophages in remyelination. Among the main proposed mechanisms are removal of myelin debris and production of growth factors known to promote oligodendrocyte differentiation such as Insulin like Growth Factor (IGF)-1 and TGF-β1. However, recent studies by Kotter et al. in demyelinating models suggest that failure in remyelination is mainly attributed to the loss of macrophages-derived promoting factors rather than their role in myelin clearance since this function can be covered by the resident microglia.

42Altogether, evidence indicates that the type of immune response is a determining factor that can promote or inhibit remyelination in demyelinating CNS lesions (as reviewed by; ). Accordingly, targeted immunomodulatory strategies rather than complete anti-inflammatory treatments appears to be a more effective strategy for promoting remyelination in autoimmune demyelinating conditions such as MS. Further research was needed to elucidate the mechanisms involved in immune response after demyelinating CNS conditions and the factors that promote remyelination.

43Following SCI or demyelination, endogenous SCs invade the injury site and contribute to remyelinating the demyelinated axons. SCs entry to the injured spinal cord occurs during the first week of injury in parallel to clearance of myelin and glial debris by microglia/macrophages. SCs enter through dorsal funiculi via dorsal root entry zone or lateral funiculi from the rootlets that become adhered to the lateral spinal cord after injury. In chemical models of demyelination in rodents, remyelination by endogenous SCs and oligodendrocytes progress simultaneously and fully myelinate demyelinated axons by 4 weeks following the insult. However, the extent of oligodendrocytes remyelination is smaller when compare to peripheral myelin formed by SCs and is restricted to the edges of the lesion. The limited degree of oligodendrocyte remyelination has been attributed to the absence of astrocytes in chemically demyelinated lesions, as oligodendrocytes are dependent on astrocytes for remyelination.

44Entry of SCs to the injured spinal cord is normally limited by glia limitans formed by astrocytes. Following insult, SCs invade the spinal cord through the regions where glia limitans is disrupted. When the glial limitans is re-established by astrocytes, SC invasion becomes progressively limited. Interestingly, studies have shown that oligodendrocytes gradually replace SCs in remyelinated axons and the transition from peripheral to central myelination occurs without any loss of function. However, other studies showed that SCs persist even chronically following SCI and continue myelinating axons. Subsequent studies investigated the transition from SCs to oligodendrocytes remyelination and found no change in SC myelination despite increasing oligodendrocyte myelination in EB and radiation (X-EB) demyelination model. There is also evidence that transplantation of OECs, SCs, and bone marrow stromal cells can promote migration of SCs from dorsal roots into the injury site. Therefore; enhanced remyelination or other beneficial effects observed after cell transplantation can be partially attributed to migrating SCs particularly in studies with poor survival of transplanted cells. This evidence suggests that SCs serve as emergency responders and protect demyelinated spinal cord axons at the time when oligodendrocytes are unable to remyelinate efficiently.

45Over the past decade, efforts from our group and others have been made to enhance oligodendrocyte replacement after SCI by cell transplantation or activating endogenous stem/progenitor cells. Cell transplantation in particular has shown promising results in enhancing SCI repair through multiple mechanisms including cell replacement, trophic support, immunomodulation, and remyelination. Using different cell types (Table 1), these studies have suggested that remyelination is a key mechanism in promoting functional recovery following SCI and demyelinating conditions.

46Potential of transplanting NPCs or glial progenitor cells in promoting remyelination has been explored in a wide variety of pathological conditions such as SCI, genetically myelin deficient rodent models, and MS. These studies have collectively demonstrated the ability of transplanted NPCs to differentiate into myelinating oligodendrocytes and ensheath demyelinated axons. Our studies in mutant Shiverer mice and rat SCI revealed that NPC-derived oligodendrocytes integrate with demyelinated and dysmyelinated axons and successfully remyelinate them (Figure 2). When we transplanted brain-derived NPCs into the spinal cord of subacutely injured rats, we found that survival and oligodendrocyte differentiation of NPCs was limited in the injury microenvironment Improving the microenvironment of engrafted NPCs with a cocktail of growth factors (EGF, bFGF, PDGF-AA) considerably promoted their long-term survival, tissue integration, and oligodendrocyte differentiation and remyelination. Importantly, in adult Shiverer mice transplanted with NPC, we found evidence of myelination and normal reconstruction of the node of Ranvier in chronically dysmyelinated axons. In the areas that NPC-derived oligodendrocytes enwrapped and myelinated the Shiverer axons, restoration of a normal configuration of paranodal and juxtaparanodal structures was achieved accompanied by improved axonal function in myelinated axons (Figure 2). Similarly, in our rat SCI studies, evidence of NPC-derived remyelination was confirmed with immunoelectron microscopy against YFP expression in transplanted YFP-NPCs. Of note, in these studies, transplantation of NPCs resulted in improved locomotor recovery evident by significant improvements in BBB and grid walking test as well as foot print analysis.

47Subsequent studies by demonstrated similar outcomes following transplantation of human glial progenitor cells into the brain of immune-deficient neonatal Shiverer mice. In this study, transplanted cells successfully differentiated into myelinating oligodendrocyte and functionally myelinated the dysmyelinated host axons in forebrain and brainstem. In agreement with our studies, immunohistological and electrophysiological evidence revealed reconstruction of the node of Ranvier in transplanted neonatal Shiverer mice and restoration of transcallosal conduction velocity. Moreover, transplanted mice showed increased lifespan and decreased seizure rate, which is frequently seen in Shiverer mice. Collectively, these studies provided proof-of-concept evidence that NPC-derived oligodendrocytes can functionally remyelinate chronically demyelinated axons in SCI and demyelinating lesions.

48Recent studies have provided further evidence that implicates remyelination as a key mechanism for neurological improvement observed after transplantation of NPCs in models of SCI. transplanted Shiverer-derived NPCs that lack the capacity for myelination into the injured spinal cord of NOD/SCID immune-deficient mice. Neuroanatomical, functional, and electrophysiological analyses demonstrated better outcomes in the injured mice transplanted with wild-type NPCs compared to the mice that received Shiverer NPCs. This work and similar study by suggest that remyelination is a key mechanism by which NPCs contributes to the functional recovery following transplantation in SCI.

49As mentioned earlier, in the post-SCI microenvironment, transplanted stem cells exhibit limited capacity for survival and migration and they primarily differentiate into astrocytes at the expense of oligodendrocytes and neurons. Genetic modifications in NPCs have been made to induce expression of specific classes of transcription or growth factors in order to enhance their survival and differentiation or modulate the hostile microenvironment of SCI. Using this approach, impact of oligodendrocyte transcription factor, Olig2 has been investigated in myelin physiology and repair. Olig2 is known to play important role in oligodendrocyte differentiation during development. Using a contusive model of rat SCI, demonstrated that retroviral overexpression of Olig2 in transplanted F3 human NPCs enhanced oligodendrocyte replacement accompanied by a significant increase in white matter sparing, decreased lesion size, increased myelin thickness and improved locomotor recovery compared to non-transduced F3 NPCs. In another study, using transgenic mice overexpressing Olig2 in SOX10+ oligodendrocytes, confirmed that Olig2 overexpression enhanced OPC differentiation, maturation, and migration as well as remyelination following chemical demyelination. Studying demyelinated lesions in human MS brain samples revealed that Olig2+ remyelinating oligodendrocytes are more abundantly present in active lesion borders compared to chronic plaques. This evidence identifies a role for Olig2 in promoting oligodendrocyte differentiation and remyelination.

50A neuroprotective role for ciliary neurotrophic factor (CNTF) in enhancing oligodendrocyte differentiation and maturation has been established in the CNS and PNS. Genetic modifications in adult rat spinal cord OPCs to express CNTF attenuated oligodendrocyte apoptosis resulting in improved survival, maturation and myelinating capacity of OPCs. CNTF expressing OPCs transplanted rats also showed significant improvements in their axonal signal conduction and hind limb locomotor recovery compared to the group that received normal OPCs.

51Altogether, current evidence suggests that NPCs can be engineered to act as environmental modulators in addition to their role in cell replacement. This strategy presents a therapeutic avenue for improving microenvironment and optimizing the outcome of cell transplantation in CNS trauma and demyelinating diseases.

52Due to challenges with oligodendrocyte differentiation of NPCs, transplantation of differentiated glial-restricted precursors (GRP) and OPCs has been pursued in SCI and demyelinating conditions. Early studies demonstrated the potential of bi-potential O2A progenitor cells for differentiation into oligodendrocytes or type 2 astrocytes. When postnatal O2A progenitor cells, genetically modified to express β-galactosidase, were transplanted into the spinal cord following x-ray induced demyelination, they showed the capacity to form oligodendrocytes and successfully remyelinated axons in demyelinated lesion. Interestingly, no evidence of astrocyte differentiation was observed after transplantation of O2A progenitors suggesting the influence of demyelinated host tissue on cell fate specification of transplanted cells. Successful remyelination also achieved following transplantation of brain-derived mouse GRPs into the spinal cord of mice with virally induced demyelination lesion that was associated with improved functional recovery.

53Application of OPCs has also shown promising results in supporting remyelination. The potential of human ESC derived OPCs for remyelination has been evaluated in animal models of SCI and demyelination. These studies uncovered that subacute stage of injury is the optimal time window for OPCs transplantation in the context of traumatic SCI. compared remyelination and functional recovery following transplantation of hESC derived OPCs at 7 days (subacute) and 10 months (chronic) after contusive SCI in rats. Subacute transplantation was associated with successful remyelination by transplanted OPCs and functional recovery while chronic transplantation did not improve remyelination. In this study, contribution of invading SCs to remyelination was also assessed. The number of SC-remyelinated axons was significantly increased in injured animals and decreased to normal levels after transplantation of hESC derived OPC. In order to quantify the contribution of SCs and OPCs in remyelination, authors relied on the difference in the thickness of myelin to differentiate between central vs peripheral myelination. However, use of immunological labeling for SCs or ultrastructural criteria of SC myelination would be a more precise approach to determine the contribution of SCs in spinal cord remyelination after injury. In addition to SCs, endogenous OPCs and NPCs also contribute to remyelination which was not investigated in this study.

54One important outcome of the Keirstead study was the inability of transplanted OPCs to remyelinate chronically demyelinated axons despite their survival and the capacity to differentiate into oligodendrocytes. This evidence suggests the inherited inhibitory nature of chronic lesions for axonal ensheathment and remyelination. Interestingly, our groups observed the same outcomes after transplantation of NPCs in chronic SCI. Our subsequent studies revealed that dense deposits of CSPGs in the chronic glial scar is a potent limiting factor to survival, integration, oligodendrocyte differentiation, and remyelination of both endogenous and transplanted NPCs. As described in earlier sections, removal of CSPGs by ChABC allowed NPC-mediated remyelination of chronically demyelinated spinal cord axons. These studies suggest that successful application of OPCs or NPCs in chronic SCI requires multifaceted interventions to modulate the inhibitory milieu of the established glial scar. Although other factors may play a role in this inhibition, CSPGs seems to be a major obstacle.

55Efficacy of transplanted OPCs has also been investigated in chronic demyelinating conditions. Transplantation of mouse ESC derived OPCs into a radiation induced rat model of cervical spinal cord demyelination 4 months after radiation therapy, showed successful survival, and migration of these cells toward the lesion and their capacity for oligodendrocyte remyelination. Moreover, significant myelin and tissue preservation as well as improved forelimb locomotor function were observed in OPC transplanted animals. Transplantation of hESC derived OPCs transplanted into athymic nude rat model of radiation induced brain demyelination, promoted remyelination of the demyelinated brain and cerebellum and ameliorated cognitive and motor deficits in the injured animals, with no evidence of tumor formation following transplantation. Absence of neuronal differentiation and insignificant astrocyte differentiation in transplanted cells indicates a defining role for remyelination by oligodendrocytes in white matter repair and cognitive and motor improvement.

56In addition to their role in replacing remyelinating oligodendrocytes, OPCs are known to enhance axo-neuronal growth and survival by producing growth factors such as brain derived neurotrophic factor (BDNF), IGF-1, glial derived growth factor (GDNF), neuregulins (NRGs), and neurotrophins that potentially contributed to the functional improvement observed in these studies. Based on current evidence, transplanted OPCs or NPCs have additional benefits beyond their known role in cell replacement and myelination including improving the host environment with providing trophic support and their immunomodulatory effects.

57Olfactory ensheathing cells (OECs) have been extensively examined for their potential for remyelination after SCI and demyelinating lesions. OECs are supporting cells that ensheath the axons of olfactory neurons. They have become a popular choice for cell transplantation due to their accessibility. Unlike oligodendrocytes or SCs, OECs do not normally myelinate axons in the nervous system. However, evidence suggests that following transplantation into the injured CNS, OECs demonstrate the ability to remyelinate axons and can promote functional recovery in animal models of SCI.

58Studies by showed evidence of remyelination by transplanted rat OECs in a rat model of X-EB induced demyelination in the thoracic spinal cord. Unlike chemical demyelination, X-EB models allow long lasting demyelination due to elimination of oligodendrocytes and astrocytes in the lesion area; therefore, excluding the potential contribution of endogenous myelinating cells. In this model, remyelination by transplanted OECs was investigated after acute and subacute OECs transplantation. Engrafted OECs were shown to integrate with the host tissue and remyelinated axons with predominant characteristic of P0 peripheral myelination. Immunoelectron microscopy at 2 and 3 weeks following transplantation confirmed OECs-derived remyelination of host demyelinated axons and reconstruction of the normal organization of the nodes of Ranvier. Functionality of OEC remyelination was confirmed by improved conduction velocity through electrophysiological recording of the spinal cords at 3 weeks after transplantation. While considerable myelination was observed following both acute and subacute lesions, the extent of myelination was considerably higher in acutely transplanted group.

59Another study in nonhuman primate demonstrated the ability of OECs to remyelinate spinal cord axons following demyelination lesions. In this study, porcine OECs were transplanted into chemically induced demyelinated adult female African green monkey spinal cord 3 days after the induction of lesion. Evidence of remyelination was observed at 3-5 weeks after transplantation in 62.5% of the transplant recipients. Endogenously remyelinated axons were evident in the vicinity of OECs-myelinated axons in the lesion while no remyelination was observed in non-transplanted group at this time-point. However, this study was not able to clearly distinguish between OECs and SCs derived remyelination.

60Since SCs and OECs demonstrate similar characteristics, their behavior and response have been compared in demyelinated spinal cord in co-transplantation studies. Interestingly, these studies suggest distinct migratory and proliferative properties for OECs compared to SCs. injected a mixture of SCs and OECs at 1 week after X irradiation into the rat spinal cord. Only transplanted OECs migrated through white and gray matter of the irradiated spinal cord. Four weeks after transplantation, a new EB demyelination was induced in the spinal cord. Tissue analysis revealed that “pre-loaded” OECs but not SCs were able to migrate toward the new EB lesion and populate the demyelinated site. Subsequent studies by the same induced demyelination using X irradiation in the juvenile rats hippocampus confirmed a better migratory ability for OECs in populating the lesion compared to SCs similar to the pattern observed in the adult host.

61While extensive evidence suggests a direct role for OECs in axonal ensheathing and remyelinating, there are also some reports that have questioned their myelinating capability in SCI., using Lacz expressing OECs, were able to track transplanted OECs in the spinal cord of injured rats. While labeled OECs were evident in the lesion at 3 weeks post-transplantation, electron micrographs of the injured spinal cord showed no direct evidence of axonal ensheathment and myelination by LacZ labeled OECs. Transplanted LacZ labeled OECs mainly enveloped a group of axons myelinated by SCs. The authors concluded that OECs support SCI repair by other mechanisms such as providing permissive substrate for axon growth and SC remyelination. In contrast to these observations, another study using superparamagnetic iron oxide labeling demonstrated the ability of both transplanted SCs and OECs for remyelination in X-EB spinal cord demyelination. Subsequently, transplanted GFP expressing OECs into the injured sciatic nerve and using immunoelectron microscopy showed that transplanted GFP+ OECs formed peripheral type myelin around axons. However, one question that remained undetermined in these studies was the degree of SC contamination in their OEC culture. Degree of SCs contamination in OEC cultures is an important consideration that may underlie different outcomes seen in these studies.

62Regardless of their direct role in axonal remyelination, OECS are shown to recondition injury environment by producing a host of trophic factors including NGF, BDNF, and CNTF that can support endogenous repair. Studies have shown that transplanted OECs enhance functional recovery through enhanced angiogenesis and immunomodulatory effects at acute and subacute stages post-transplantation. demonstrated that transplantation of OECs results in upregulation of Cox-2 and vascular endothelial growth factor (VEGF) following SCI.

63Collectively, while application of OECs has shown promising results in experimental models of SCI and demyelination, the ability of OECs for CNS remyelination is still a matter of debate due to their similarities with SCs and potential contamination of OEC cultures with SCs. Further in vitro and in vivo studies using specific markers of OECs and SCs is required to specifically distinguish these two cell populations and confirm the myelinating capacity of OECs.

64While cell translation is a promising approach for enhancing remyelination following injury, graft rejection due to the host immune reaction poses a major challenge to the success of cell-based therapies. Successful transplantation in animal models has required immunosuppression, wherein chance of the graft rejection had been minimized using immunosuppressive therapy or genetically immune-deficient models. Importantly, immune reaction is a hallmark of pathologic CNS that plays essential roles in tissue injury and repair. Hence, use of autologous sources of stem cells seems to be a logical solution to warrant the long-term survival of transplanted cells without compromising the host CNS immune response. Moreover, autologous transplantation preclude ethical and technical limitations in using embryonic stem cells or fetal derived tissues that has been a major hurdle in translating this promising repair strategy into clinical arenas.

65Generation of induced pluripotent stem cells (iPSCs) using autologous somatic cells has opened new avenues for developing clinically feasible cell transplantation approaches. Since the introduction of iPSCs technology in stem cell research, several studies have explored the therapeutic potential of transplanting iPSCs-derived NPCs or OPCs in SCI for inducing remyelination as well as the safety of this approach with regards to tumorigenesis. transplanted allogenic NPCs generated from mouse embryonic fibroblast (MEF)-derived iPSCs into a mouse model of contusive SCI at 9 days post injury. The safety of this approach was previously confirmed by observing no teratoma formation after 24 weeks of transplantation into NOD/SCID mouse brain. Six weeks following transplantation, MEF-iPSC derived NPCs showed multilineage capacity and differentiated into oligodendrocytes, astrocytes, and neurons. Functional analysis suggested improved neurological recovery in the transplanted animals, which was accompanied by enhanced remyelination as well as regeneration of the raphe-spinal fibers in transplanted animals. Similar observations have been made using a variety of adult tissue derived iPSCs. Transplantation of selected non-tumorigenic clone of adult tail tip fibroblast derived iPSCs into a mouse model of contusive SCI resulted in significant functional recovery. In mouse and primate models of SCI, transplanted NPCs derived from human adult facial skin derived iPSCs were able to differentiate into neurons, oligodendrocytes, and astrocytes which resulted in enhanced functional recovery accompanied by axonal growth.

66Remyelination has been considered as a mechanism underlying the functional improvement observed in iPSCs transplantation strategies. A recent study by demonstrated that transplanted OPCs generated from autologous skin derived human iPSCs of keratinocyte and fibroblast origin, were able to differentiate into mature myelinating oligodendrocytes and myelinate human fetal cortical neurons in vitro. In immune-deficient Shiverer mice, human iPSC-derived OPCs integrated with the host neonatal brain tissue after transplantation, differentiated to MBP expressing oligodendrocytes, remyelinated the dysmyelinated Shiverer axons, and constructed the nodes of Ranvier. No tumorigenicity was observed in this study and transplanted Shiverer mice exhibited increased lifespan.

67Taken together, emerging evidence shows the potential and feasibility of transplanting iPSCs derived NPCs and OPCs for promoting oligodendrocyte replacement and remyelination in demyelinating conditions such as MS and traumatic CNS injury. While promising, the risk of tumorigenicity and proper cell differentiation of this therapy have yet remained to be carefully investigated. In Table 1, we have listed cell therapies that have been developed to promote oligodendrocytes replacement and remyelination after SCI and MS conditions.

68The myelin sheath is an essential component in the CNS and PNS that ensures rapid signal transduction in axons through the nervous system. Myelinated fibers show a highly specialized and organized structure at the node of Ranvier that is vital to their proper functioning. Damage to oligodendrocytes due to trauma or disease results in demyelination that causes aberrant localization and expression of ion channels associated with axonal dysfunction. Although considerable remyelination occurs spontaneously by endogenous CNS progenitor cells, the quality and extend of myelination is not optimal to restore structural and physiological properties of injured axons. Emerging research evidence has uncovered several mechanisms by which oligodendrocyte differentiation and remyelination are regulated within the microenvironment of injury. Inhibitory role of activated glial cells and insufficient expression of promoting factors are major limiting factors that impede remyelination. Over the past years, extensive research efforts have been made to therapeutically promote remyelination following CNS trauma or demyelinating disease. Outcomes of the recent pharmacological and cell-based therapies indicate the impact of remyelination as a mechanism for improving function after injury. Further investigations are needed to develop effective and feasible repair strategies with potential for clinical translation.

69The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.