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Objective: Transplanted neural stem/precursor cells (NPCs) display peculiar therapeutic plasticity in vivo. Although the replacement of cells was first expected as the prime therapeutic mechanism of stem cells in regenerative medicine, it is now clear that transplanted NPCs simultaneously instruct several therapeutic mechanisms, among which replacement of cells might not necessarily prevail. A comprehensive understanding of the mechanism(s) by which NPCs exert their therapeutic plasticity is lacking. This study was designed as a preclinical approach to test the feasibility of human NPC transplantation in an outbreed nonhuman primate experimental autoimmune encephalomyelitis (EAE) model approximating the clinical and complex neuropathological situation of human multiple sclerosis (MS) more closely than EAE in the standard laboratory rodent. Methods: We examined the safety and efficacy of the intravenous (IV) and intrathecal (IT) administration of human NPCs in common marmosets affected by human myelin oligodendrocyte glycoprotein 1-125–induced EAE. Treatment commenced upon the occurrence of detectable brain lesions on a 4.7T spectrometer. Results: EAE marmosets injected IV or IT with NPCs accumulated lower disability and displayed increased survival, as compared with sham-treated controls. Transplanted NPCs persisted within the host central nervous system (CNS), but were also found in draining lymph nodes, for up to 3 months after transplantation and exhibited remarkable immune regulatory capacity in vitro. Interpretation: Herein, we provide the first evidence that human CNS stem cells ameliorate EAE in nonhuman primates without overt side effects. Immune regulation (rather than neural differentiation) is suggested as the major putative mechanism by which NPCs ameliorate EAE in vivo. Our findings represent a critical step toward the clinical use of human NPCs in MS. Ann Neurol 2009;66:343–354

Endogenous neural stem/precursor cells (NPCs) are considered a functional reservoir for promoting tissue homeostasis and repair after injury, therefore regenerative strategies that mobilize these cells have recently been proposed. Despite evidence of increased neurogenesis upon acute inflammatory insults (e.g. ischaemic stroke), the plasticity of the endogenous brain stem cell compartment in chronic CNS inflammatory disorders remains poorly characterized. Here we show that persistent brain inflammation, induced by immune cells targeting myelin, extensively alters the proliferative and migratory properties of subventricular zone (SVZ)-resident NPCs in vivo leading to significant accumulation of non-migratory neuroblasts within the SVZ germinal niche. In parallel, we demonstrate a quantitative reduction of the putative brain stem cells proliferation in the SVZ during persistent brain inflammation, which is completely reversed after in vitro culture of the isolated NPCs. Together, these data indicate that the inflamed brain microenvironment sustains a non cell-autonomous dysfunction of the endogenous CNS stem cell compartment and challenge the potential efficacy of proposed therapies aimed at mobilizing endogenous precursors in chronic inflammatory brain disorders.

Adult multipotent neural stem/precursor cells (NPCs) have the capacity to self-renew and generate functional differentiated cells (e.g. neurons, astrocytes or oligodendrocytes) within discrete tissue-specific germinal niches, such as the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus of the hippocampus. Due to their intrinsic plasticity NPCs can be considered an essential part of the cellular mechanism(s) by which the central nervous system (CNS) tries to repair itself after an injury and, as a consequence, they also represents an attractive therapeutic tool for the treatment of neurological disorders. Here we will discuss not only the role of NPC-based transplantation therapies in multiple sclerosis (MS) but also recent data suggesting that endogenous NPCs, while contributing to CNS repair in MS, may also become the target of the disease itself.

Eliciting the in situ accumulation and persistence patterns of stem cells following transplantation would provide critical insight toward human translation of stem cell-based therapies. To this end, we have developed a strategy to track neural stem/precursor cells (NPCs) in vivo using magnetic resonance (MR) imaging. Initially, we evaluated three different human-grade superparamagnetic iron oxide particles for labeling NPCs and found the optimal labeling to be achieved with Resovist. Next, we carried out in vivo experiments to monitor the accumulation of Resovist-labeled NPCs following i.v. injection in mice with experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. With a human MR scanner, we were able to visualize transplanted cells as early as 24 hours post-transplantation in up to 80% of the brain demyelinating lesions. Interestingly, continued monitoring of transplanted mice indicated that labeled NPCs were still present 20 days postinjection. Neuropathological analysis confirmed the presence of transplanted NPCs exclusively in inflammatory demyelinating lesions and not in normal-appearing brain areas. Quantification of transplanted cells by means of MR-based ex vivo relaxometry (R2*) showed significantly higher R2* values in focal inflammatory brain lesions from EAE mice transplanted with labeled NPCs as compared with controls. Indeed, sensitive quantification of low numbers of NPCs accumulating into brain inflammatory lesions (33.3–164.4 cells per lesion; r2 = .998) was also obtained. These studies provide evidence that clinical-grade human MR can be used for noninvasive monitoring and quantification of NPC accumulation in the central nervous system upon systemic cell injection. Disclosure of potential conflicts of interest is found at the end of this article.

UNLABELLED: Cortical reorganization occurring in multiple sclerosis (MS) patients is thought to play a key role in limiting the effect of structural tissue damage. Conversely, its exhaustion may contribute to the irreversible disability that accumulates with disease progression. Several aspects of MS-related cortical reorganization, including the overall functional effect and likely modulation by therapies, still remain to be elucidated. The aim of this work was to assess the extent of functional cortical reorganization and its brain structural/pathological correlates in Dark Agouti rats with experimental autoimmune encephalomyelitis (EAE), a widely accepted preclinical model of chronic MS. Morphological and functional MRI (fMRI) were performed before disease induction and during the relapsing and chronic phases of EAE. During somatosensory stimulation of the right forepaw, fMRI demonstrated that cortical reorganization occurs in both relapsing and chronic phases of EAE with increased activated volume and decreased laterality index versus baseline values. Voxel-based morphometry demonstrated gray matter (GM) atrophy in the cerebral cortex, and both GM and white matter atrophy were assessed by ex vivo pathology of the sensorimotor cortex and corpus callosum. Neuroinflammation persisted in the relapsing and chronic phases, with dendritic spine density in the layer IV sensory neurons inversely correlating with the number of cluster of differentiation 45-positive inflammatory lesions. Our work provides an innovative experimental platform that may be pivotal for the comprehension of key mechanisms responsible for the accumulation of irreversible brain damage and for the development of innovative therapies to reduce disability in EAE/MS. SIGNIFICANCE STATEMENT: Since the early 2000s, functional MRI (fMRI) has demonstrated profound modifications in the recruitment of cortical areas during motor, cognitive, and sensory tasks in multiple sclerosis (MS) patients. Experimental autoimmune encephalomyelitis (EAE) represents a reliable model of the chronic-progressive variant of MS. fMRI studies in EAE have not been performed extensively up to now. This paper reports fMRI studies in a rat model of MS with somatosensory stimulation of the forepaw. We demonstrated modifications in the recruitment of cortical areas consistent with data from MS patients. To the best of our knowledge, this is the first report of cortical remodeling in a preclinical in vivo model of MS.

We tested cortical motor evoked potentials (cMEPs) as a quantitative marker for in vivo monitoring of corticospinal tract damage in a murine multiple sclerosis model (experimental autoimmune encephalomyelitis, EAE). The cMEPs, previously standardized in naive C57BL/6 developing and adult mice, were studied longitudinally in adult EAE mice. Central conduction times (CCTs) increased significantly shortly before the earliest clinical signs developed (10 days postimmunization, dpi), with peak delay in acute EAE (20-40 dpi). In clinically stable disease (80 dpi), CCTs did not increase further, but cMEP amplitude declined progressively, with complete loss in >80% of mice at 120 dpi. Increase in CCT correlated with presence of inflammatory infiltrates and demyelination in acute EAE, whereas small or absent cMEPs were associated with continuing axonal damage in clinically-stabilized disease and beyond (>80 dpi). These results demonstrate that cMEPs are a useful method for monitoring corticospinal tract function in chronic-progressive EAE, and provide insight into the pathological substrate of the condition.

Gene therapy protocols aimed to deliver therapeutic molecules into the central nervous system may represent an alternative therapeutic strategy in patients affected by inflammatory demyelinating diseases of the central nervous system where systemic therapies have shown limited therapeutic efficacy possibly owing to the blood-brain barrier, a major obstacle for the entry of therapeutic molecules into the central nervous system.Among inflammatory demyelinating diseases of the central nervous system, gene therapy approaches have been so far developed almost exclusively for multiple sclerosis. However, the chronic/relapsing nature of the disease, the restriction to the central nervous system of the pathological process as well as the necessity to inhibit the ongoing inflammatory process but also to foster endogenous remyelinating pathways, have posed several questions which still need to be properly addressed for the development of a successful gene therapy strategy in multiple sclerosis patients.The gene therapy approaches for multiple sclerosis have been so far developed and tested only in rodents and monkeys with experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. The results of these studies clearly indicate that the delivery of therapeutic genes within the central nervous system is superior to the peripheral delivery. In particular, the intracerebral delivery of genes coding for anti-inflammatory and/or neurotrophic molecules, using gene vectors derived from non-replicative viruses, showed to inhibit not only the detrimental function of blood-borne mononuclear effector cells but also to foster proliferation and differentiation of surviving oligodendrocytes within demyelinated areas. Here, we summarize the most recent findings of this novel area of research.

Polyreactive (auto)antibodies are frequently found in healthy subjects and are bona fide considered to be nonpathogenic. However, autoreactive B cells and circulating (auto)antibodies have been associated with several neurological syndromes, including demyelinating disorders. Whether these antibodies can have a real impact on disease development is still a matter of debate. Here, we briefly summarize some of the most recently published data on both the deleterious and the protective effects of antibodies in autoimmune demyelinating disorders of the central nervous system.

Physiological (spontaneous) and reactive (reparative) regenerative processes are fundamental part of life and greatly differ among the different animals and tissues. While spontaneous regeneration naturally occurs upon cell attrition, reparative regeneration occurs as a consequence of tissue damage. Both spontaneous and reparative regeneration play an important role in maintaining the normal equilibrium of the central nervous system (CNS) as well as in promoting its repair upon injury. Cells play a critical role in reparative regeneration as regenerating structures (cells or tissues) depend on the proliferation without (de)differentiation of parenchymal cells surviving to the injury, proliferation of stem (progenitor) cells resident in the injured tissue, dedifferentiation of mature cells in the remaining tissue, or by the influx of stem cells originating outside the damaged tissue. Considering the central role of stem and progenitor cells in regeneration, a spur of experimental stem cell-based transplantation approaches for tissue (e.g. CNS) repair has been recently generated. This review will focus on the therapeutic efficacy of different sources of somatic stem cells - and in particular on those of neural origin - in promoting CNS repair in a chronic (auto)immune-mediated inflammatory disorder such as multiple sclerosis.

Central nervous system (CNS) delivery of anti-inflammatory cytokines, such as interleukin 4 (IL4), holds promise as treatment for multiple sclerosis (MS). We have previously shown that short-term herpes simplex virus type 1-mediated IL4 gene therapy is able to inhibit experimental autoimmune encephalomyelitis (EAE), an animal model of MS, in mice and non-human primates. Here, we show that a single administration of an IL4-expressing helper-dependent adenoviral vector (HD-Ad) into the cerebrospinal fluid (CSF) circulation of immunocompetent mice allows persistent transduction of neuroepithelial cells and long-term (up to 5 months) CNS transgene expression without toxicity. Mice affected by chronic and relapsing EAE display clinical and neurophysiological recovery from the disease once injected with the IL4-expressing HD-Ad vector. The therapeutic effect is due to the ability of IL4 to increase, in inflamed CNS areas, chemokines (CCL1, CCL17 and CCL22) capable of recruiting regulatory T cells (CD4+CD69- CD25+Foxp3+) with suppressant functions. CSF delivery of HD-Ad vectors expressing anti-inflammatory molecules might represent a valuable therapeutic option for CNS inflammatory disorders.

Spontaneous remyelination occurs in multiple sclerosis (MS) patients. However, this process is not robust enough to promote a functional and stable recovery of the myelin architecture in demyelinated areas of the central nervous system (CNS). As a consequence of this incomplete reparative process, the disease invariably progresses and patchy areas of demyelination-in which axonal damage and/or loss is a constant accompanying factor-increase over time and lead to the accumulation of irreversible neurological deficits. Thus, the development of cell-based therapies aimed to promote multifocal remyelination in MS represents one of the most challenging areas of investigation. Several cell-replacement strategies have been developed in the last few years. However, most of these therapeutic approaches-although consistently able to form new myelin sheaths around the transplantation site-are unrealistic owing to the multifocality of the demyelinating process and the inability to in vitro growth and differentiate large number of myelin-forming cells. Recently, promising cell-replacement therapies based on the use of stem cells have been proposed. However, before envisaging any potential human applications of such therapies we need to confront with some preliminary and still unsolved questions: (i) the ideal stem cell source for transplantation, (ii) the route of cell administration, (iii) the differentiation and persistence of stem cells into the targeted tissue and, last but not least, (iv) the functional and long-lasting integration of transplanted cells into the host tissue.

Selective interference with immune processes in the central nervous system (CNS) is a very difficult task because of the limitations associated with the delivery of immuno modulatory molecules across the blood brain barrier. Systemic administration of immune-mediators, either by conventional routes or by intramuscularly or intravenous gene therapy, is hampered by severe side effects and alters immune-system functions also in peripheral organs. To overcome these problems, different gene therapy strategies have been developed to deliver immuno modulatory molecules directly within the central nervous system. The use of engineered CNS antigen-specific circulating cells as selective delivery vehicles, the direct injection of gene vectors into the brain parenchyma, or also the ependymal route, have been proposed as possible alternative gene therapy protocols to selectively interfere with immuno-pathological processes in the CNS. We will review the use of these CNS-targeted gene therapy protocols for the treatment of experimental autoimmune encephalomyelitis (EAE), the prototypical experimental immune-mediated disease of the CNS, and therefore discuss the relevance of these results for the therapy of multiple sclerosis (MS) the most common, immune-mediated, demyelinating disease of the CNS in humans.

Widespread demyelination and axonal loss are the pathological hallmarks of multiple sclerosis. The multifocal nature of this chronic inflammatory disease of the central nervous system complicates cellular therapy and puts emphasis on both the donor cell origin and the route of cell transplantation. We established syngenic adult neural stem cell cultures and injected them into an animal model of multiple sclerosis--experimental autoimmune encephalomyelitis (EAE) in the mouse--either intravenously or intracerebroventricularly. In both cases, significant numbers of donor cells entered into demyelinating areas of the central nervous system and differentiated into mature brain cells. Within these areas, oligodendrocyte progenitors markedly increased, with many of them being of donor origin and actively remyelinating axons. Furthermore, a significant reduction of astrogliosis and a marked decrease in the extent of demyelination and axonal loss were observed in transplanted animals. The functional impairment caused by EAE was almost abolished in transplanted mice, both clinically and neurophysiologically. Thus, adult neural precursor cells promote multifocal remyelination and functional recovery after intravenous or intrathecal injection in a chronic model of multiple sclerosis.