Morito Takano

Significance of Remyelination by Neural Stem/Progenitor Cells Transplanted into the Injured Spinal Cord†‡§

Previous reports of functional recovery from spinal cord injury (SCI) in rodents and monkeys after the delayed transplantation of neural stem/progenitor cells (NS/PCs) have raised hopes that stem cell therapy could be used to treat SCI in humans. More research is needed, however, to understand the mechanism of functional recovery. Oligodendrocytes derived from grafted NS/PCs remyelinate spared axons in the injured spinal cord. Here, we studied the extent of this remyelinations contribution to functional recovery following contusive SCI in mice. To isolate the effect of remyelination from other possible regenerative benefits of the grafted cells, NS/PCs obtained from myelin‐deficient shiverer mutant mice (shi‐NS/PCs) were used in this work alongside wild‐type NS/PCs (wt‐NS/PCs). shi‐NS/PCs behaved like wt‐NS/PCs in vitro and in vivo, with the exception of their myelinating potential. shi‐NS/PC‐derived oligodendrocytes did not express myelin basic protein in vitro and formed much thinner myelin sheaths in vivo compared with wt‐NS/PC‐derived oligodendrocytes. The transplantation of shi‐NS/PCs promoted some locomotor and electrophysiological functional recovery but significantly less than that afforded by wt‐NS/PCs. These findings establish the biological importance of remyelination by graft‐derived cells for functional recovery after the transplantation of NS/PCs into the injured spinal cord. STEM CELLS 2011;29:1983–1994.

Neural stem cells directly differentiated from partially reprogrammed fibroblasts rapidly acquire gliogenic competency

Neural stem cells (NSCs) were directly induced from mouse fibroblasts using four reprogramming factors (Oct4, Sox2, Klf4, and cMyc) without the clonal isolation of induced pluripotent stem cells (iPSCs). These NSCs gave rise to both neurons and glial cells even at early passages, while early NSCs derived from clonal embryonic stem cells (ESCs)/iPSCs differentiated mainly into neurons. Epidermal growth factor‐dependent neurosphere cultivation efficiently propagated these gliogenic NSCs and eliminated residual pluripotent cells that could form teratomas in vivo. We concluded that these directly induced NSCs were derived from partially reprogrammed cells, because dissociated ESCs/iPSCs did not form neurospheres in this culture condition. These NSCs differentiated into both neurons and glial cells in vivo after being transplanted intracranially into mouse striatum. NSCs could also be directly induced from adult human fibroblasts. The direct differentiation of partially reprogrammed cells may be useful for rapidly preparing NSCs with a strongly reduced propensity for tumorigenesis. STEM CELLS2012;30:1109–1119

Time-dependent changes in the microenvironment of injured spinal cord affects the therapeutic potential of neural stem cell transplantation for spinal cord injury

BackgroundThe transplantation of neural stem/progenitor cells (NS/PCs) at the sub-acute phase of spinal cord injury, but not at the chronic phase, can promote functional recovery. However, the reasons for this difference and whether it involves the survival and/or fate of grafted cells under these two conditions remain unclear. To address this question, NS/PC transplantation was performed after contusive spinal cord injury in adult mice at the sub-acute and chronic phases.ResultsQuantitative analyses using bio-imaging, which can noninvasively detect surviving grafted cells in living animals, revealed no significant difference in the survival rate of grafted cells between the sub-acute and chronic transplantation groups. Additionally, immunohistology revealed no significant difference in the differentiation phenotypes of grafted cells between the two groups. Microarray analysis revealed no significant differences in the expression of genes encoding inflammatory cytokines or growth factors, which affect the survival and/or fate of grafted cells, in the injured spinal cord between the sub-acute and chronic phases. By contrast, the distribution of chronically grafted NS/PCs was restricted compared to NS/PCs grafted at the sub-acute phase because a more prominent glial scar located around the lesion epicenter enclosed the grafted cells. Furthermore, microarray and histological analysis revealed that the infiltration of macrophages, especially M2 macrophages, which have anti-inflammatory role, was significantly higher at the sub-acute phase than the chronic phase. Ultimately, NS/PCs that were transplanted in the sub-acute phase, but not the chronic phase, promoted functional recovery compared with the vehicle control group.ConclusionsThe extent of glial scar formation and the characteristics of inflammation is the most remarkable difference in the injured spinal cord microenvironment between the sub-acute and chronic phases. To achieve functional recovery by NS/PC transplantation in cases at the chronic phase, modification of the microenvironment of the injured spinal cord focusing on glial scar formation and inflammatory phenotype should be considered.

Grafted Human iPS Cell-Derived Oligodendrocyte Precursor Cells Contribute to Robust Remyelination of Demyelinated Axons after Spinal Cord Injury

Summary Murine- and human-induced pluripotent stem cell-derived neural stem/progenitor cells (iPSC-NS/PCs) promote functional recovery following transplantation into the injured spinal cord in rodents and primates. Although remyelination of spared demyelinated axons is a critical mechanism in the regeneration of the injured spinal cord, human iPSC-NS/PCs predominantly differentiate into neurons both in vitro and in vivo. We therefore took advantage of our recently developed protocol to obtain human-induced pluripotent stem cell-derived oligodendrocyte precursor cell-enriched neural stem/progenitor cells and report the benefits of transplanting these cells in a spinal cord injury (SCI) model. We describe how this approach contributes to the robust remyelination of demyelinated axons and facilitates functional recovery after SCI.

Transplantation of neural stem/progenitor cells at different locations in mice with spinal cord injury

Transplantation of neural stem/progenitor cells (NS/PCs) promotes functional recovery after spinal cord injury (SCI); however, few studies have examined the optimal site of NS/PC transplantation in the spinal cord. The purpose of this study was to determine the optimal transplantation site of NS/PCs for the treatment of SCI. Wild-type mice were generated with contusive SCI at the T10 level, and NS/PCs were derived from fetal transgenic mice. These NS/PCs ubiquitously expressed ffLuc-cp156 protein (Venus and luciferase fusion protein) and so could be detected by in vivo bioluminescence imaging 9 days postinjury. NS/PCs (low: 250,000 cells per mouse; high: 1 million cells per mouse) were grafted into the spinal cord at the lesion epicenter (E) or at rostral and caudal (RC) sites. Phosphate-buffered saline was injected into E as a control. Motor functional recovery was better in each of the transplantation groups (E-Low, E-High, RC-Low, and RC-High) than in the control group. The photon counts of the grafted NS/PCs were similar in each of the four transplantation groups, suggesting that the survival of NS/PCs was fairly uniform when more than a certain threshold number of cells were transplanted. Quantitative RT-PCR analyses demonstrated that brain-derived neurotropic factor expression was higher in the RC segment than in the E segment, and this may underlie why NS/PCs more readily differentiated into neurons than into astrocytes in the RC group. The location of the transplantation site did not affect the area of spared fibers, angiogenesis, or the expression of any other mediators. These findings indicated that the microenvironments of the E and RC sites are able to support NS/PCs transplanted during the subacute phase of SCI similarly. Optimally, a certain threshold number of NS/PCs should be grafted into the E segment to avoid damaging sites adjacent to the lesion during the injection procedure.

Controlling Immune Rejection Is a Fail-Safe System against Potential Tumorigenicity after Human iPSC-Derived Neural Stem Cell Transplantation

Our previous work reported functional recovery after transplantation of mouse and human induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NS/PCs) into rodent models of spinal cord injury (SCI). Although hiPSC-NS/PCs proved useful for the treatment of SCI, the tumorigenicity of the transplanted cells must be resolved before they can be used in clinical applications. The current study sought to determine the feasibility of ablation of the tumors formed after hiPSC-NS/PC transplantation through immunoregulation. Tumorigenic hiPSC-NS/PCs were transplanted into the intact spinal cords of immunocompetent BALB/cA mice with or without immunosuppressant treatment. In vivo bioluminescence imaging was used to evaluate the chronological survival and growth of the transplanted cells. The graft survival rate was 0% in the group without immunosuppressants versus 100% in the group with immunosuppressants. Most of the mice that received immunosuppressants exhibited hind-limb paralysis owing to tumor growth at 3 months after iPSC-NS/PC transplantation. Histological analysis showed that the tumors shared certain characteristics with low-grade gliomas rather than with teratomas. After confirming the progression of the tumors in immunosuppressed mice, the immunosuppressant agents were discontinued, resulting in the complete rejection of iPSC-NS/PC-derived masses within 42 days after drug cessation. In accordance with the tumor rejection, hind-limb motor function was recovered in all of the mice. Moreover, infiltration of microglia and lymphocytes was observed during the course of tumor rejection, along with apoptosis of iPSC-NS/PC-generated cells. Thus, immune rejection can be used as a fail-safe system against potential tumorigenicity after transplantation of iPSC-NS/PCs to treat SCI.

Inflammatory cascades mediate synapse elimination in spinal cord compression

BackgroundCervical compressive myelopathy (CCM) is caused by chronic spinal cord compression due to spondylosis, a degenerative disc disease, and ossification of the ligaments. Tip-toe walking Yoshimura (twy) mice are reported to be an ideal animal model for CCM-related neuronal dysfunction, because they develop spontaneous spinal cord compression without any artificial manipulation. Previous histological studies showed that neurons are lost due to apoptosis in CCM, but the mechanism underlying this neurodegeneration was not fully elucidated. The purpose of this study was to investigate the pathophysiology of CCM by evaluating the global gene expression of the compressed spinal cord and comparing the transcriptome analysis with the physical and histological findings in twy mice.MethodsTwenty-week-old twy mice were divided into two groups according to the magnetic resonance imaging (MRI) findings: a severe compression (S) group and a mild compression (M) group. The transcriptome was analyzed by microarray and RT-PCR. The cellular pathophysiology was examined by immunohistological analysis and immuno-electron microscopy. Motor function was assessed by Rotarod treadmill latency and stride-length tests.ResultsSevere cervical calcification caused spinal canal stenosis and low functional capacity in twy mice. The microarray analysis revealed 215 genes that showed significantly different expression levels between the S and the M groups. Pathway analysis revealed that genes expressed at higher levels in the S group were enriched for terms related to the regulation of inflammation in the compressed spinal cord. M1 macrophage-dominant inflammation was present in the S group, and cysteine-rich protein 61 (Cyr61), an inducer of M1 macrophages, was markedly upregulated in these spinal cords. Furthermore, C1q, which initiates the classical complement cascade, was more upregulated in the S group than in the M group. The confocal and electron microscopy observations indicated that classically activated microglia/macrophages had migrated to the compressed spinal cord and eliminated synaptic terminals.ConclusionsWe revealed the detailed pathophysiology of the inflammatory response in an animal model of chronic spinal cord compression. Our findings suggest that complement-mediated synapse elimination is a central mechanism underlying the neurodegeneration in CCM.

MRI Characterization of Paranodal Junction Failure and Related Spinal Cord Changes in Mice

The paranodal junction is a specialized axon-glia contact zone that is important for normal neuronal activity and behavioral locomotor function in the central nervous system (CNS). Histological examination has been the only method for detecting pathological paranodal junction conditions. Recently, diffusion tensor MRI (DTI) has been used to detect microstructural changes in various CNS diseases. This study was conducted to determine whether MRI and DTI could detect structural changes in the paranodal junctions of the spinal cord in cerebroside sulfotransferase knock-out (CST-KO) mice. Here, we showed that high-resolution MRI and DTI characteristics can reflect paranodal junction failure in CST-KO mice. We found significantly lower T1 times and significantly higher T2 times in the spinal cord MRIs of CST-KO mice as compared to wild-type (WT) mice. Spinal cord DTI showed significantly lower axial diffusivity and significantly higher radial diffusivity in CST-KO mice as compared to WT mice. In contrast, the histological differences in the paranodal junctions of WT and CST-KO mice were so subtle that electron microscopy or immunohistological analyses were necessary to detect them. We also measured gait disturbance in the CST-KO mice, and determined the conduction latency by electrophysiology. These findings demonstrate the potential of using MRI and DTI to evaluate white matter disorders that involve paranodal junction failure.

Distinct roles of endogenous vascular endothelial factor receptor 1 and 2 in neural protection after spinal cord injury

Secondary degeneration after spinal cord injury (SCI) is caused by increased vascular permeability, infiltration of inflammatory cells, and subsequent focal edema. Therapeutic interventions using neurotrophic factors have focused on the prevention of such reactions to reduce cell death and promote tissue regeneration. Vascular endothelial growth factor (VEGF) is a potent angiogenic and vascular permeability factor. However, the effect of VEGF on SCI remains controversial. VEGF signaling is primarily regulated through two primary receptors, VEGF receptor 1 (VEGF-R1) and VEGF receptor 2 (VEGF-R2). The purpose of this study was to examine the effects of intraperitoneal administration of VEGF-R1- and VEGF-R2-neutralizing antibodies on a mouse model of SCI. VEGF-R1 blockade, but not VEGF-R2 blockade, decreased the permeability and infiltration of inflammatory cells, and VEGF-R2 blockade caused a significant increase in neuronal apoptosis in the acute phase of SCI. VEGF-R2 blockade decreased the residual tissue area and the number of neural fibers in the chronic phase of SCI. VEGF-R2 blockade worsened the functional recovery and prolonged the latency of motor evoked potentials. These data suggest that endogenous VEGF-R2 plays a crucial role in neuronal protection after SCI.

Enhanced Functional Recovery from Spinal Cord Injury in Aged Mice after Stem Cell Transplantation through HGF Induction

Summary The number of elderly patients with spinal cord injury (SCI) is increasing worldwide, representing a serious burden for both the affected patients and the community. Previous studies have demonstrated that neural stem cell (NSC) transplantation is an effective treatment for SCI in young animals. Here we show that NSC transplantation is as effective in aged mice as it is in young mice, even though aged mice exhibit more severe neurological deficits after SCI. NSCs grafted into aged mice exhibited better survival than those grafted into young mice. Furthermore, we show that the neurotrophic factor HGF plays a key role in the enhanced functional recovery after NSC transplantation observed in aged mice with SCI. The unexpected results of the present study suggest that NSC transplantation is a potential therapeutic modality for SCI, even in elderly patients.