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The conjugation/de-conjugation of Small Ubiquitin-like Modifier (SUMO) has been shown to be associated with a diverse set of physiologic/pathologic conditions. The clinical significance and ostensible therapeutic utility offered via the selective control of the global SUMOylation process has become readily apparent in ischemic pathophysiology. Herein, we describe the development of a novel quantitative high-throughput screening (qHTS) system designed to identify small molecules capable of increasing SUMOylation via the regulation/inhibition of members of the microRNA (miRNA)-182 family. This assay employs a SHSY5Y human neuroblastoma cell line stably transfected with a dual firefly-Renilla luciferase reporter system for identification of specific inhibitors of either miR-182 or miR-183. In this study, we have identified small molecules capable of inducing increased global conjugation of SUMO in both SHSY5Y cells and rat E18-derived primary cortical neurons. The protective effects of a number of the identified compounds were confirmed via an in vitro ischemic model (oxygen/glucose deprivation). Of note, this assay can be easily repurposed to allow high-throughput analyses of the potential drugability of other relevant miRNA(s) in ischemic pathobiology.

Compelling evidence exists that somatic neural stem/precursor cell (NPC)-based therapies protect the central nervous system (CNS) from chronic inflammation-driven degeneration, such as that occurring in experimental autoimmune encephalomyelitis (EAE), multiple sclerosis (MS), cerebral ischemic/hemorrhagic stroke and spinal cord injury (SCI). However, while it was first assumed that NPC transplants may act through direct replacement of lost/damaged cells, it has now become clear that they are able to protect the damaged nervous system through a number of 'bystander' mechanisms other than the expected cell replacement. In immune-mediated experimental demyelination--both in rodents and non-human primates--others and we have shown that transplanted NPC possess a constitutive and inducible ability to mediate efficient 'bystander' myelin repair and axonal rescue. This novel mechanism(s), which may improve the success of transplantation procedures, is likely to be exerted by undifferentiated NPCs whose functional characteristics are regulated by both CNS-resident and blood-borne inflammatory cells releasing in situ major stem cell regulators. Here, we discuss some of these alternative 'bystander' mechanisms, while pointing at the formation of the atypical ectopic perivascular niches, as the most challenging example of reciprocal biologically sound cross talk between the inflamed microenvironment(s) and transplanted therapeutic NPCs.

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

In ischemic stroke, the third most frequent cause of mortality in industrialized countries, therapeutic options have until now been limited to the first hours after disease onset. Cell transplantation has emerged in various neurological disorders, including experimental stroke, as a successful recovery-promoting approach also in the post-acute stroke phase. However, before envisaging any translation into humans of such promising cell-based approaches we still need to clarify: (i) the ideal cell source for transplantation, (ii) the most appropriate route of cell administration, and, last but not least, (iii) the best approach to achieve an appropriate and functional integration of transplanted cells into the host tissue. Here we discuss, with special emphasis on neural stem/precursor cells, potential mechanisms that may be involved in the action of cell-based therapies in stroke.

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.

Udgivelsesdato: 2009-Aug Recent evidence suggests that neural stem/precursor cells (NPCs) promote recovery in animal models with delayed neuronal death via a number of indirect bystander effects. A comprehensive knowledge of how transplanted NPCs exert their therapeutic effects is still lacking. Here, we investigated the effects of a delayed transplantation of adult syngenic NPCs--injected intravenously 72 h after transient middle cerebral artery occlusion--on neurological recovery, histopathology and gene expression. NPC-transplanted mice showed a significantly improved recovery from 18 days post-transplantation (dpt) onwards, which persisted throughout the study. A small percentage of injected NPCs accumulated in the brain, integrating mainly in the infarct boundary zone, where most of the NPCs remained undifferentiated up to 30 dpt. Histopathological analysis revealed a hitherto unreported very delayed neuroprotective effect of NPCs, becoming evident at 10 and 30 dpt. Tissue survival was associated with downregulation of markers of inflammation, glial scar formation and neuronal apoptotic death at both mRNA and protein levels. Our data highlight the relevance of very delayed degenerative processes in the stroke brain that are intimately associated with inflammatory and glial responses. These processes may efficaciously be antagonized by (stem) cell-based strategies at time-points far beyond established therapeutic windows for pharmacological neuroprotection.

Background: The systemic injection of neural stem/precursor cells (NPCs) provides remarkable amelioration of the clinicopathological features of experimental autoimmune encephalomyelitis (EAE). This is dependent on the capacity of transplanted NPCs to engage concurrent mechanisms of action within specific microenvironments in vivo. Among a wide range of therapeutic actions alternative to cell replacement, neuroprotective and immune modulatory capacities of transplanted NPCs have been described. However, lacking is a detailed understanding of the mechanisms by which NPCs exert their therapeutic plasticity. This study was designed to identify the first candidate that exemplifies and sustains the immune modulatory capacity of transplanted NPCs. Methodology/Principal Findings: To achieve the exclusive targeting of the peripheral immune system, SJL mice with PLP-induced EAE were injected subcutaneously with NPCs and the treatment commenced prior to disease onset. NPC-injected EAE mice showed significant clinical improvement, as compared to controls. Exogenous NPCs lacking the expression of major neural antigens were reliably (and for long-term) found at the level of draining lymph nodes, while establishing sophisticated anatomical interactions with lymph node cells. Importantly, injected NPCs were never found in organs other than lymph nodes, including the brain and the spinal cord. Draining lymph nodes from transplanted mice showed focal upregulation of major developmental stem cell regulators, such as BMP-4, Noggin and Sonic hedgehog. In lymph nodes, injected NPCs hampered the activation of myeloid dendritic cells (DCs) and steadily restrained the expansion of antigen-specific encephalitogenic T cells. Both ex vivo and in vitro experiments identified a novel highly NPC-specific-BMP-4-dependent-mechanism hindering the DC maturation. Conclusion/Significance: The study described herein, identifies the first member of the TGF beta/BMP family of stem cell regulators as a novel tolerogenic factor released by NPCs. Full exploitation of this pathway as an efficient tool for vaccination therapy in autoimmune inflammatory conditions is underway. Background: The systemic injection of neural stem/precursor cells (NPCs) provides remarkable amelioration of the clinicopathological features of experimental autoimmune encephalomyelitis (EAE). This is dependent on the capacity of transplanted NPCs to engage concurrent mechanisms of action within specific microenvironments in vivo. Among a wide range of therapeutic actions alternative to cell replacement, neuroprotective and immune modulatory capacities of transplanted NPCs have been described. However, lacking is a detailed understanding of the mechanisms by which NPCs exert their therapeutic plasticity. This study was designed to identify the first candidate that exemplifies and sustains the immune modulatory capacity of transplanted NPCs. Methodology/Principal Findings: To achieve the exclusive targeting of the peripheral immune system, SJL mice with PLP-induced EAE were injected subcutaneously with NPCs and the treatment commenced prior to disease onset. NPC-injected EAE mice showed significant clinical improvement, as compared to controls. Exogenous NPCs lacking the expression of major neural antigens were reliably (and for long-term) found at the level of draining lymph nodes, while establishing sophisticated anatomical interactions with lymph node cells. Importantly, injected NPCs were never found in organs other than lymph nodes, including the brain and the spinal cord. Draining lymph nodes from transplanted mice showed focal upregulation of major developmental stem cell regulators, such as BMP-4, Noggin and Sonic hedgehog. In lymph nodes, injected NPCs hampered the activation of myeloid dendritic cells (DCs) and steadily restrained the expansion of antigen-specific encephalitogenic T cells. Both ex vivo and in vitro experiments identified a novel highly NPC-specific-BMP-4-dependent-mechanism hindering the DC maturation. Conclusion/Significance: The study described herein, identifies the first member of the TGF beta/BMP family of stem cell regulators as a novel tolerogenic factor released by NPCs. Full exploitation of this pathway as an efficient tool for vaccination therapy in autoimmune inflammatory conditions is underway.

Neural stem/precursor cells (NPCs) are broadly proposed as an alternative cell source to repair brain damage upon transplantation. NPC-driven brain repair has variably been shown in several pre-clinical models of neurological disorders. However, a comprehensive knowledge of the different mechanism(s) by which such cells exert their therapeutic potential is still lacking. While the replacement of lost or damaged cells was until a few years ago assumed to be the prime therapeutic mechanism of stem cells, it is now clear that transplanted somatic stem cells may simultaneously instruct several therapeutic mechanisms not confined to cell replacement on its own. Combining the overall therapeutic potential of NPCs in neurological disease, the concept of therapeutic plasticity has recently been proposed (Martino and Pluchino, 2006). The brain repair potential of transplanted stem cells in stroke-like conditions has solid pre-clinical evidence. It has been shown, with variable results, that NPCs—transplanted either systemically or intraparenchymally—display peculiar pathotropism toward specific sites of ischaemic damage, survive within the host for long periods of time, and establish a functional cross-talk with the different cell types of the (micro)environment. This NPC-dependent operational behaviour was first demonstrated to be capable of promoting CNS tissue recovery in mice with experimental bacterial collagenase-induced intracerebral haemorrage (ICH) (Jeong et al. , 2003) or acute middle cerebral artery occlusion-induced ischaemic stroke (Chu et al. , 2004). Interestingly, transplanted NPCs promoted significant functional …

Transplantation of neural stem cells holds great promise for treating neurological disorders. Martino and Pluchino argue that neural stem cells achieve their therapeutic efficacy exculsively by a cell-replacement mechanism, rather than by the recently proposed alternative mechanism of bystander neuroprotection. Recent evidence shows that transplantation of neural stem/precursor cells may protect the central nervous system from inflammatory damage through a 'bystander' mechanism that is alternative to cell replacement. This novel mechanism, which might improve the success of transplantation procedures, is exerted by undifferentiated neural stem cells, the functional characteristics of which are regulated by important stem cell regulators released by CNS-resident and blood-borne inflammatory cells. Here, we discuss this alternative bystander mechanism in the context of the atypical ectopic perivascular niche. We propose that it is the most challenging example of reciprocal therapeutic crosstalk between the inflamed CNS and systemically transplanted neural stem cells.

The well regulated activities of microglia and T cells specific to central nervous system (CNS) antigens can contribute to the protection of CNS neural cells and their renewal from adult neural stem/progenitor cells (aNPCs). Here we report that T cell-based vaccination of mice with a myelin-derived peptide, when combined with transplantation of aNPCs into the cerebrospinal fluid (CSF), synergistically promoted functional recovery after spinal cord injury. The synergistic effect was correlated with modulation of the nature and intensity of the local T cell and microglial response, expression of brain-derived neurotrophic factor and noggin protein, and appearance of newly formed neurons from endogenous precursor-cell pools. These results substantiate the contention that the local immune response plays a crucial role in recruitment of aNPCs to the lesion site, and suggest that similar immunological manipulations might also serve as a therapeutic means for controlled migration of stem/progenitor cells to other acutely injured CNS sites.

Spontaneous neural tissue repair occurs in patients affected by inflammatory and degenerative disorders of the central nervous system (CNS). However, this process is not robust enough to promote a functional and stable recovery of the CNS architecture. The development of cell-based therapies aimed at promoting brain repair, through damaged cell-replacement, is therefore foreseen. Several experimental cell-based strategies aimed at replacing damaged neural cells have been developed in the last 30 years. Although successful in promoting site-specific repair in focal CNS disorders, most of these therapeutic approaches have failed to foster repair in multifocal CNS diseases where the anatomical and functional damage is widespread. Stem cell-based therapies have been recently proposed and might represent in the near future a plausible alternative strategy in these disorders. However, before envisaging any human applications of stem cell-based therapies in neurological diseases, we need to consider some preliminary and still unsolved issues: (i) the ideal stem cell source for transplantation, (ii) the most appropriate route of stem cell administration, and, last but not least, (iii) the best approach to achieve an appropriate, functional, and long-lasting integration of transplanted stem cells into the host tissue.

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.