Eftekhar Eftekharpour

Delayed Transplantation of Adult Neural Precursor Cells Promotes Remyelination and Functional Neurological Recovery after Spinal Cord Injury

Spinal cord injury (SCI) results in loss of oligodendrocytes demyelination of surviving axons and severe functional impairment. Spontaneous remyelination is limited. Thus, cell replacement therapy is an attractive approach for myelin repair. In this study, we transplanted adult brain-derived neural precursor cells (NPCs) isolated from yellow fluorescent protein-expressing transgenic mice into the injured spinal cord of adult rats at 2 and 8 weeks after injury, which represents the subacute and chronic phases of SCI. A combination of growth factors, the anti-inflammatory drug minocycline, and cyclosporine A immunosuppression was used to enhance the survival of transplanted adult NPCs. Our results show the presence of a substantial number of surviving NPCs in the injured spinal cord up to 10 weeks after transplantation at the subacute stage of SCI. In contrast, cell survival was poor after transplantation into chronic lesions. After subacute transplantation, grafted cells migrated >5 mm rostrally and caudally. The surviving NPCs integrated principally along white-matter tracts and displayed close contact with the host axons and glial cells. Approximately 50% of grafted cells formed either oligodendroglial precursor cells or mature oligodendrocytes. NPC-derived oligodendrocytes expressed myelin basic protein and ensheathed the axons. We also observed that injured rats receiving NPC transplants had improved functional recovery as assessed by the Basso, Beattie, and Bresnahan Locomotor Rating Scale and grid-walk and footprint analyses. Our data provide strong evidence in support of the feasibility of adult NPCs for cell-based remyelination after SCI.

Synergistic effects of transplanted adult neural stem/progenitor cells, chondroitinase, and growth factors promote functional repair and plasticity of the chronically injured spinal cord.

The transplantation of neural stem/progenitor cells (NPCs) is a promising therapeutic strategy for spinal cord injury (SCI). However, to date NPC transplantation has exhibited only limited success in the treatment of chronic SCI. Here, we show that chondroitin sulfate proteoglycans (CSPGs) in the glial scar around the site of chronic SCI negatively influence the long-term survival and integration of transplanted NPCs and their therapeutic potential for promoting functional repair and plasticity. We targeted CSPGs in the chronically injured spinal cord by sustained infusion of chondroitinase ABC (ChABC). One week later, the same rats were treated with transplants of NPCs and transient infusion of growth factors, EGF, bFGF, and PDGF-AA. We demonstrate that perturbing CSPGs dramatically optimizes NPC transplantation in chronic SCI. Engrafted NPCs successfully integrate and extensively migrate within the host spinal cord and principally differentiate into oligodendrocytes. Furthermore, this combined strategy promoted the axonal integrity and plasticity of the corticospinal tract and enhanced the plasticity of descending serotonergic pathways. These neuroanatomical changes were also associated with significantly improved neurobehavioral recovery after chronic SCI. Importantly, this strategy did not enhance the aberrant synaptic connectivity of pain afferents, nor did it exacerbate posttraumatic neuropathic pain. For the first time, we demonstrate key biological and functional benefits for the combined use of ChABC, growth factors, and NPCs to repair the chronically injured spinal cord. These findings could potentially bring us closer to the application of NPCs for patients suffering from chronic SCI or other conditions characterized by the formation of a glial scar.

Current status of experimental cell replacement approaches to spinal cord injury

✓ Despite advances in medical and surgical care, the current clinical therapies for spinal cord injury (SCI) are largely ineffective. During the last 2 decades, the search for new therapies has been revolutionized by the discovery of stem cells, which has inspired scientists and clinicians to search for a stem cell–based reparative approaches to many diseases, including neurotrauma. In the present study, the authors briefly summarize current knowledge related to the pathophysiology of SCI, including the concepts of primary and secondary injury and the importance of posttraumatic demyelination. Key inhibitory obstacles that impede axonal regeneration include the glial scar and a number of myelin inhibitory molecules including Nogo. Recent advancements in cell replacement therapy as a therapeutic strategy for SCI are summarized. The strategies include the use of pluripotent human stem cells, embryonic stem cells, and a number of adult-derived stem and progenitor cells such as mesenchymal stem cells, Schwann...Despite advances in medical and surgical care, the current clinical therapies for spinal cord injury (SCI) are largely ineffective. During the last 2 decades, the search for new therapies has been revolutionized by the discovery of stem cells, which has inspired scientists and clinicians to search for a stem cell-based reparative approaches to many diseases, including neurotrauma. In the present study, the authors briefly summarize current knowledge related to the pathophysiology of SCI, including the concepts of primary and secondary injury and the importance of posttraumatic demyelination. Key inhibitory obstacles that impede axonal regeneration include the glial scar and a number of myelin inhibitory molecules including Nogo. Recent advancements in cell replacement therapy as a therapeutic strategy for SCI are summarized. The strategies include the use of pluripotent human stem cells, embryonic stem cells, and a number of adult-derived stem and progenitor cells such as mesenchymal stem cells, Schwann cells, olfactory ensheathing cells, and adult-derived neural precursor cells. Although current strategies to repair the subacutely injured cord appear promising, many obstacles continue to render the treatment of chronic injuries challenging. Nonetheless, the future for stem cell-based reparative strategies for treating SCI appears bright.

Myelination of Congenitally Dysmyelinated Spinal Cord Axons by Adult Neural Precursor Cells Results in Formation of Nodes of Ranvier and Improved Axonal Conduction

Emerging evidence suggests that cell-based remyelination strategies may be a feasible therapeutic approach for CNS diseases characterized by myelin deficiency as a result of trauma, congenital anomalies, or diseases. Although experimental demyelination models targeted at the transient elimination of oligodendrocytes have suggested that transplantation-based remyelination can partially restore axonal molecular structure and function, it is not clear whether such therapeutic approaches can be used to achieve functional remyelination in models associated with long-term, irreversible myelin deficiency. In this study, we transplanted adult neural precursor cells (aNPCs) from the brain of adult transgenic mice into the spinal cords of adult Shiverer (shi/shi) mice, which lack compact CNS myelin. Six weeks after transplantation, the transplanted aNPCs expressed oligodendrocyte markers, including MBP, migrated extensively along the white matter tracts of the spinal cord, and formed compact myelin. Conventional and three-dimensional confocal and electron microscopy revealed axonal ensheathment, establishment of paranodal junctional complexes leading to de novo formation of nodes of Ranvier, and partial reconstruction of the juxtaparanodal and paranodal molecular regions of axons based on Kv1.2 and Caspr (contactin-associated protein) expression by the transplanted aNPCs. Electrophysiological recordings revealed improved axonal conduction along the transplanted segments of spinal cords. We conclude that myelination of congenitally dysmyelinated adult CNS axons by grafted aNPCs results in the formation of compact myelin, reconstruction of nodes of Ranvier, and enhanced axonal conduction. These data suggest the therapeutic potential of aNPCs to promote functionally significant myelination in CNS disorders characterized by longstanding myelin deficiency.

Temporal and spatial patterns of Kv1.1 and Kv1.2 protein and gene expression in spinal cord white matter after acute and chronic spinal cord injury in rats: implications for axonal pathophysiology after neurotrauma

After spinal cord injury (SCI), surviving white matter axons display axonal dysfunction associated with demyelination and altered K+ channel activity. To clarify the molecular basis of posttraumatic axonal pathophysiology after SCI, we investigated the changes in expression and distribution of the axonal K+ channel subunits Kv1.1 and Kv1.2 in spinal cord white matter after in vivo SCI in the rat. Using Western blot analysis, we found an increased expression of Kv1.1 and Kv1.2 at 2 and 6 weeks after SCI. By real‐time PCR we observed an increase in Kv1.1 and Kv1.2 mRNA levels 1 day after SCI, which persisted until 6 weeks. Confocal immunohistochemistry showed a markedly dispersed labelling of Kv1.1 and Kv1.2 along the injured axons, in contrast to the tight localization of these channels to the juxtaparanodes of noninjured axons. This redistribution of Kv1.1 and Kv1.2 occurred as early as 1 h postinjury along some injured axons, and persisted at 6 weeks postinjury. In parallel with the redistribution of Kv1.1 and 1.2, contactin‐associated protein (Caspr), which is normally confined to a paranodal location, also displayed a more diffuse distribution along the injured spinal cord axons. Our results suggest that the increased expression of Kv1.1 and Kv1.2 proteins is transcriptionally regulated. In contrast, the redistribution of the axonal K+ channel subunits occurs very early postinjury and probably reflects a disruption of the juxtaparanodal axonal region due to physical trauma, as shown by altered localization of Caspr.

Are Induced Pluripotent Stem Cells the Future of Cell-Based Regenerative Therapies for Spinal Cord Injury?

Despite advances in medical and surgical care, current clinical therapies for spinal cord injury (SCI) are limited. During the last two decades, the search for new therapies has been revolutionized by the discovery of stem cells, inspiring scientists and clinicians to search for stem cell‐based reparative approaches for many disorders, including neurotrauma. Cell‐based therapies using embryonic and adult stem cells in animal models of these disorders have provided positive outcome results. However, the availability of clinically suitable cell sources for human application has been hindered by both technical and ethical issues. The recent discovery of induced pluripotent stem (iPS) cells holds the potential to revolutionize the field of regenerative medicine by offering the option of autologous transplantation, thus eliminating the issue of host rejection. Herein, we will provide the rationale for the use of iPS cells in SCI therapies. In this review, we will evaluate the recent advancements in the field of iPS cells including their capacity for differentiation toward neural lineages that may allow iPS cells transplantation in cell‐based therapy for spinal cord repair. J. Cell. Physiol. 222: 515–521, 2010.

Structural and functional alterations of spinal cord axons in adult Long Evans Shaker (LES) dysmyelinated rats.

Abnormal formation or loss of myelin is a distinguishing feature of many neurological disorders and contributes to the pathobiology of neurotrauma. In this study we characterize the functional and molecular changes in CNS white matter in Long Evans Shaker (LES) rats. These rats have a spontaneous mutation of the gene encoding myelin basic protein which results in severe dysmyelination of the central nervous system (CNS), providing a unique model for demyelinating/dysmyelinating disorders. To date, the functional and molecular changes in CNS white matter in this model are not well understood. We have used in vivo somatosensory evoked potential (SSEP), in vitro compound action potential (CAP) recording in isolated dorsal columns, confocal immunohistochemistry, Western blotting and real-time PCR to examine the electrophysiological, molecular and cellular changes in spinal cord white matter in LES rats. We observed that dysmyelination is associated with dispersed labeling of Kv1.1 and Kv1.2 K+ channel subunits, as well as Caspr, a protein normally confined to paranodes, along the LES rat spinal cord axons. Abnormal electrophysiological properties including attenuation of CAP amplitude and conduction velocity, high frequency conduction failure and enhanced sensitivity to K+ channel blockers 4-aminopyridine and dendrotoxin-I were observed in spinal cord axons from LES rats. Our results in LES rats clarify some of the key molecular, cellular and functional consequences of dysmyelination and myelin-axon interactions. Further understanding of these issues in this model could provide critical insights for neurological disorders characterized by demyelination.

Stem cells and spinal cord injury repair.

Spinal cord injury (SCI) has remained a challenging area for scientists and clinicians due to the adverse and complex nature of its pathobiology. To date, clinical therapies for debilitating SCI are largely ineffective. However, emerging research evidence suggests that repair of SCI can be promoted by stem cell-based therapies in regenerative medicine. Over the past decade, therapeutic potential of different types of stem cells for the treatment of SCI have been investigated in preclinical models. These studies have revealed multiple beneficial roles by which stem cells can improve the outcomes of SCI. This chapter will summarize the recent advances in the application of stem cells in regenerative medicine for the repair of SCI.

Generation of neural stem cells from embryonic stem cells using the default mechanism: in vitro and in vivo characterization.

Neural stem cell-based approaches to repair damaged white matter in the central nervous system have shown great promise; however, the optimal cell population to employ in these therapies remains undetermined. A default mechanism of neural induction may function during development, and in embryonic stem cells (ESCs) neural differentiation is elicited in the absence of any extrinsic signaling in minimal, serum-free culture conditions. The default mechanism can be used to derive clonal neurosphere-forming populations of neural stem cells that have been termed leukemia inhibitory factor-dependent primitive neural stem cells (pNSCs), which subsequently give rise to fibroblast growth factor 2-dependent definitive NSCs (dNSCs). Here we characterized the neural differentiation pattern of these two cell types in vitro and in vivo when transplanted into the dysmyelinated spinal cords of shiverer mice. We compared the differentiation pattern to that observed for neural stem/progenitor cells derived from the adult forebrain subependymal zone [adult neural precursor cells (aNPCs)]. dNSCs produced a differentiation pattern similar to that of aNPCs in vitro and in the shiverer model in vivo, where both cell types produced terminally differentiated oligodendrocytes that associated with host axons and expressed myelin basic protein. This is the first demonstration of the in vivo differentiation of NSCs, derived from ESCs through the default mechanism, into the oligodendrocyte lineage. We conclude that dNSCs derived through the default pathway of neural induction are a similar cell population to aNPCs and that the default mechanism is a promising approach to generate NSCs from pluripotent cell populations for use in cell therapy or other research applications.

Genome-wide gene expression profiling of stress response in a spinal cord clip compression injury model

BackgroundThe aneurysm clip impact-compression model of spinal cord injury (SCI) is a standard injury model in animals that closely mimics the primary mechanism of most human injuries: acute impact and persisting compression. Its histo-pathological and behavioural outcomes are extensively similar to human SCI. To understand the distinct molecular events underlying this injury model we analyzed global mRNA abundance changes during the acute, subacute and chronic stages of a moderate to severe injury to the rat spinal cord.ResultsTime-series expression analyses resulted in clustering of the majority of deregulated transcripts into eight statistically significant expression profiles. Systematic application of Gene Ontology (GO) enrichment pathway analysis allowed inference of biological processes participating in SCI pathology. Temporal analysis identified events specific to and common between acute, subacute and chronic time-points. Processes common to all phases of injury include blood coagulation, cellular extravasation, leukocyte cell-cell adhesion, the integrin-mediated signaling pathway, cytokine production and secretion, neutrophil chemotaxis, phagocytosis, response to hypoxia and reactive oxygen species, angiogenesis, apoptosis, inflammatory processes and ossification. Importantly, various elements of adaptive and induced innate immune responses span, not only the acute and subacute phases, but also persist throughout the chronic phase of SCI. Induced innate responses, such as Toll-like receptor signaling, are more active during the acute phase but persist throughout the chronic phase. However, adaptive immune response processes such as B and T cell activation, proliferation, and migration, T cell differentiation, B and T cell receptor-mediated signaling, and B cell- and immunoglobulin-mediated immune response become more significant during the chronic phase.ConclusionsThis analysis showed that, surprisingly, the diverse series of molecular events that occur in the acute and subacute stages persist into the chronic stage of SCI. The strong agreement between our results and previous findings suggest that our analytical approach will be useful in revealing other biological processes and genes contributing to SCI pathology.