Differential and cooperative actions of Olig1 and Olig2 transcription factors on immature proliferating cells after contusive spinal cord injury.

Spontaneous remyelination after spinal cord injury (SCI) is limited probably due to inadequate signaling to generate sufficient OLs from progenitor cells. The present study tested a hypothesis that introduction of olig genes, critical regulators of OL development, into immature proliferating cells could increase oligodendrogenesis after contusive SCI in adult rats. Recombinant retroviruses encoding Olig1 and Olig2 transcription factors, separately or in combination, with green fluorescent protein (GFP) were injected into the injured spinal cord. Unexpectedly, introduction of Olig2-GFP retroviruses led to a marked hyperplasia of GFP+ cells at 1 week, and soft agar colony forming assay of isolated GFP+ cells confirmed Olig2-induced tumorous transformation. In contrast, Olig1 did not alter the number of GFP+ cells. Simultaneous expression of Olig1 and Olig2 (Olig1/2) led to a marked increase in the number of GFP+ cells without tumor formation. The proportion of GFP+ cells with OL progenitor markers was increased by Olig1/2. Moreover, Olig1/2 robustly increased the proportion of mature OLs and expression of myelin related proteins, while Olig1 alone exhibited only modest effects. Olig1/2 upregulated Sox10, which drives terminal OL differentiation, implicating Sox 10 as a mediator of Olig1/2 effects on the maturation. Finally, injection of Olig1/2 retroviruses significantly improved a quality of hindpaws locomotion and increased the total number of OLs after SCI. Activation of both Olig1 and Olig2 may be beneficial by both increasing the progenitor cell proliferation and enhancing OL differentiation in the injured spinal cord.

Overexpression of Bcl-XL in human neural stem cells promotes graft survival and functional recovery following transplantation in spinal cord injury.

Transplantation of neural stem cells (NSCs) has shown promise for improving functional recovery after spinal cord injury (SCI). The inhospitable milieu of injured spinal cord, however, does not support survival of grafted NSCs, reducing therapeutic efficacy of transplantation. The present study sought to examine whether overexpression of antiapoptotic gene Bcl-X(L) in NSCs could promote graft survival and functional recovery following transplantation in rat contusive SCI model. A human NSC line (HB1.F3) was transduced with a retroviral vector encoding Bcl-X(L) to generate Bcl-X(L)-overexpressing NSCs (HB1.F3.Bcl-X(L)). Overexpression of Bcl-X(L) conferred resistance to staurosporine-mediated apoptosis. The number of HB1.F3.Bcl-X(L) cells was 1.5-fold higher at 2 weeks and 10-fold higher at 7 weeks posttransplantation than that of HB1.F3 cells. There was no decline in the number of HB1.F3.Bcl-X(L) cells between 2 and 7 weeks, indicating that Bcl-X(L) overexpression completely blocked cell death occurring between these two time points. Transplantation of HB1.F3.Bcl-X(L) cells improved locomotor scores and enhanced accuracy of hindlimb placement in a grid walk. Approximately 10% of surviving NSCs differentiated into oligodendrocytes. Surviving NSCs produced brain-derived neurotrophic factor (BDNF), and the level of BDNF was significantly increased only in the HB1.F3.Bcl-X(L) group. Transplantation of HB1.F3.Bcl-X(L) cells reduced cavity volumes and enhanced white matter sparing. Finally, HB1.F3.Bcl-X(L) grafts enhanced connectivity between the red nucleus and the spinal cord below the lesion. These results suggest that enhancing graft survival with antiapoptotic gene can potentiate therapeutic benefits of NSC-based therapy for SCI.

Transplantation of human neural stem cells transduced with Olig2 transcription factor improves locomotor recovery and enhances myelination in the white matter of rat spinal cord following contusive injury.

BACKGROUND: Contusive spinal cord injury is complicated by a delayed loss of oligodendrocytes, resulting in chronic progressive demyelination. Therefore, transplantation strategies to provide oligodendrocyte lineage cells and to enhance the extent of myelination appear to be justified for spinal cord repair. The present study investigated whether transplantation of human neural stem cells (NSCs) genetically modified to express Olig2 transcription factor, an essential regulator of oligodendrocyte development, can improve locomotor recovery and enhance myelination in a rat contusive spinal cord injury model. RESULTS: HB1.F3 (F3) immortalized human NSC line was transduced with a retroviral vector encoding Olig2, an essential regulator of oligodendrocyte development. Overexpression of Olig2 in human NSCs (F3.Olig2) induced activation of NKX2.2 and directed differentiation of NSCs into oligodendrocyte lineage cells in vitro. Introduction of Olig2 conferred higher proliferative activity, and a much larger number of F3.Olig2 NSCs were detected by 7 weeks after transplantation into contused spinal cord than that of parental F3 NSCs. F3.Olig2 NSCs exhibited frequent migration towards the white matter, whereas F3 NSCs were mostly confined to the gray matter or around the lesion cavities. Most of F3.Olig2 NSCs occupying the spared white matter differentiated into mature oligodendrocytes. Transplantation of F3.Olig2 NSCs increased the volume of spared white matter and reduced the cavity volume. Moreover, F3.Olig2 grafts significantly increased the thickness of myelin sheath around the axons in the spared white matter. Finally, animals with F3.Olig2 grafts showed an improvement in the quality of hindlimbs locomotion. CONCLUSION: Transplantation of NSCs genetically modified to differentiate into an oligodendrocytic lineage may be an effective strategy to improve functional outcomes following spinal cord trauma. The present study suggests that molecular factors governing cell fate decisions can be manipulated to enhance reparative potential of the cell-based therapy.

Gene transfer mediated by stem cell grafts to treat CNS injury.

INTRODUCTION: Stem cell transplantation holds promise for promoting anatomical repair and functional recovery after traumatic or ischemic injuries to the CNS. Harnessing stem cells with therapeutic genes of interest is regarded as an attractive approach to augment therapeutic benefits of stem cell grafts. AREAS COVERED: The advantage of stem-cell-mediated gene transfer is the engraftibility of stem cells that can ensure a long-term and stable expression of therapeutic genes. In addition, stem-cell-gene interaction may synergistically amplify therapeutic benefits. Delivery of classical neurotrophic factor genes provided neuroprotective and pro-regenerative effects in various injury models. Some studies employed therapeutic genes targeting post-injury microenvironment to support endogenous repair. Recent trials of stem-cell-mediated transfer of nonclassical growth factors showed relatively novel biological effects. Combinatorial strategies seem to have the potential to improve therapeutic efficacy. EXPERT OPINION: Future development of induced pluripotent stem cells and novel scaffolding biomaterials will greatly expedite the advances in ex vivo gene therapy to treat CNS injury. Before moving to a clinical stage, rigorous preclinical evaluations are needed to identify an optimal gene or gene combination in different injury settings. Improving the safety of viral vectors will be a critical prerequisite for the clinical translation.

Combination of multifaceted strategies to maximize the therapeutic benefits of neural stem cell transplantation for spinal cord repair.

Neural stem cells (NSCs) possess therapeutic potentials to reverse complex pathological processes following spinal cord injury (SCI), but many obstacles remain that could not be fully overcome by NSC transplantation alone. Combining complementary strategies might be required to advance NSC-based treatments to the clinical stage. The present study was undertaken to examine whether combination of NSCs, polymer scaffolds, neurotrophin-3 (NT3), and chondroitinase, which cleaves chondroitin sulfate proteoglycans at the interface between spinal cord and implanted scaffold, could provide additive therapeutic benefits. In a rat hemisection model, poly(ɛ-caprolactone) (PCL) was used as a bridging scaffold and as a vehicle for NSC delivery. The PCL scaffolds seeded with F3 NSCs or NT3 overexpressing F3 cells (F3.NT3) were implanted into hemisected cavities. F3.NT3 showed better survival and migration, and more frequently differentiated into neurons and oligodendrocytes than F3 cells. Animals with PCL scaffold containing F3.NT3 cells showed the best locomotor recovery, and motor evoked potentials (MEPs) following transcranial magnetic stimulation were recorded only in PCL-F3.NT3 group in contralateral, but not ipsilateral, hindlimbs. Implantation of PCL scaffold with F3.NT3 cells increased NT3 levels, promoted neuroplasticity, and enhanced remyelination of contralateral white matter. Combining chondroitinase treatment after PCL-F3.NT3 implantation further enhanced cell migration and promoted axonal remodeling, and this was accompanied by augmented locomotor recovery and restoration of MEPs in ipsilateral hindlimbs. We demonstrate that combining multifaceted strategies can maximize the therapeutic benefits of NSC transplantation for SCI. Our results may have important clinical implications for the design of future NSC-based strategies.