Soya Kawabata

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.

Surgical correction of severe cervical kyphosis in patients with neurofibromatosis Type 1

Severe cervical kyphosis requiring surgical treatment is rare in patients with neurofibromatosis Type 1 (NF1). When it occurs, however, dystrophic changes in the vertebrae make surgical correction and fusion of the deformity extremely difficult. The authors report on 3 cases of severe cervical kyphosis associated with NF1 that were successfully treated with combined anterior and posterior correction and fusion. All patients underwent halo-gravity traction for approximately 1 month prior to surgery to correct the deformity gradually. Posterior correction and fusion were performed with segmental spinal instrumentation consisting of lateral mass screws, lamina screws, pedicle screws, and polyethylene tape for sublaminar wiring. Anterior spinal fusion was performed using a fibula strut to induce solid bone fusion. All patients used a halo vest for postoperative external fixation. Preoperative CT scans showed dystrophic cervical spine changes, and MR images demonstrated extensive neurofibromas outside the cervical spine in all 3 patients. The preoperative kyphotic angles were as follows: Case 1, 140°; Case 2, 81°; and Case 3, 72°; after halo-gravity traction, the kyphosis angles improved to 50°, 55°, and 51°, respectively; and after surgery, they were 50°, 15°, and 27°, respectively. Solid bone union was observed in all patients at the latest follow-up. All three patients experienced postoperative complications consisting of superficial infection, severe pneumonia, and partial dislocation of the distal fibula graft after removing the halo vest, in one patient each. Although dystrophic cervical vertebral changes in these patients with NF1 complicated the correction of severe cervical kyphosis, the use of preoperative halo-gravity traction, a combination of spinal instrumentations, an anterior strut bone graft, and postoperative halo-vest fixation made it possible to correct the kyphosis, maintain the correction, and achieve solid bone fusion.

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.

Fail-Safe System against Potential Tumorigenicity after Transplantation of iPSC Derivatives

Summary Human induced pluripotent stem cells (iPSCs) are promising in regenerative medicine. However, the risks of teratoma formation and the overgrowth of the transplanted cells continue to be major hurdles that must be overcome. Here, we examined the efficacy of the inducible caspase-9 (iCaspase9) gene as a fail-safe against undesired tumorigenic transformation of iPSC-derived somatic cells. We used a lentiviral vector to transduce iCaspase9 into two iPSC lines and assessed its efficacy in vitro and in vivo. In vitro, the iCaspase9 system induced apoptosis in approximately 95% of both iPSCs and iPSC-derived neural stem/progenitor cells (iPSC-NS/PCs). To determine in vivo function, we transplanted iPSC-NS/PCs into the injured spinal cord of NOD/SCID mice. All transplanted cells whose mass effect was hindering motor function recovery were ablated upon transduction of iCaspase9. Our results suggest that the iCaspase9 system may serve as an important countermeasure against post-transplantation adverse events in stem cell transplant therapies.

Pretreatment with a γ-Secretase Inhibitor Prevents Tumor-like Overgrowth in Human iPSC-Derived Transplants for Spinal Cord Injury

Summary Neural stem/progenitor cells (NS/PCs) derived from human induced pluripotent stem cells (hiPSCs) are considered to be a promising cell source for cell-based interventions that target CNS disorders. We previously reported that transplanting certain hiPSC-NS/PCs in the spinal cord results in tumor-like overgrowth of hiPSC-NS/PCs and subsequent deterioration of motor function. Remnant immature cells should be removed or induced into more mature cell types to avoid adverse effects of hiPSC-NS/PC transplantation. Because Notch signaling plays a role in maintaining NS/PCs, we evaluated the effects of γ-secretase inhibitor (GSI) and found that pretreating hiPSC-NS/PCs with GSI promoted neuronal differentiation and maturation in vitro, and GSI pretreatment also reduced the overgrowth of transplanted hiPSC-NS/PCs and inhibited the deterioration of motor function in vivo. These results indicate that pretreatment with hiPSC-NS/PCs decreases the proliferative capacity of transplanted hiPSC-NS/PCs, triggers neuronal commitment, and improves the safety of hiPSC-based approaches in regenerative medicine.

Regulation of RhoA by STAT3 coordinates glial scar formation

Understanding how the transcription factor signal transducer and activator of transcription–3 (STAT3) controls glial scar formation may have important clinical implications. We show that astrocytic STAT3 is associated with greater amounts of secreted MMP2, a crucial protease in scar formation. Moreover, we report that STAT3 inhibits the small GTPase RhoA and thereby controls actomyosin tonus, adhesion turnover, and migration of reactive astrocytes, as well as corralling of leukocytes in vitro. The inhibition of RhoA by STAT3 involves ezrin, the phosphorylation of which is reduced in STAT3-CKO astrocytes. Reduction of phosphatase and tensin homologue (PTEN) levels in STAT3-CKO rescues reactive astrocytes dynamics in vitro. By specific targeting of lesion-proximal, reactive astrocytes in Nestin-Cre mice, we show that reduction of PTEN rescues glial scar formation in Nestin-Stat3+/− mice. These findings reveal novel intracellular signaling mechanisms underlying the contribution of reactive astrocyte dynamics to glial scar formation.

Pathological classification of human iPSC-derived neural stem/progenitor cells towards safety assessment of transplantation therapy for CNS diseases

The risk of tumorigenicity is a hurdle for regenerative medicine using induced pluripotent stem cells (iPSCs). Although teratoma formation is readily distinguishable, the malignant transformation of iPSC derivatives has not been clearly defined due to insufficient analysis of histology and phenotype. In the present study, we evaluated the histology of neural stem/progenitor cells (NSPCs) generated from integration-free human peripheral blood mononuclear cell (PBMC)-derived iPSCs (iPSC-NSPCs) following transplantation into central nervous system (CNS) of immunodeficient mice. We found that transplanted iPSC-NSPCs produced differentiation patterns resembling those in embryonic CNS development, and that the microenvironment of the final site of migration affected their maturational stage. Genomic instability of iPSCs correlated with increased proliferation of transplants, although no carcinogenesis was evident. The histological classifications presented here may provide cues for addressing potential safety issues confronting regenerative medicine involving iPSCs.

Safe and efficient method for cryopreservation of human induced pluripotent stem cell-derived neural stem and progenitor cells by a programmed freezer with a magnetic field.

Stem cells represent a potential cellular resource in the development of regenerative medicine approaches to the treatment of pathologies in which specific cells are degenerated or damaged by genetic abnormality, disease, or injury. Securing sufficient supplies of cells suited to the demands of cell transplantation, however, remains challenging, and the establishment of safe and efficient cell banking procedures is an important goal. Cryopreservation allows the storage of stem cells for prolonged time periods while maintaining them in adequate condition for use in clinical settings. Conventional cryopreservation systems include slow-freezing and vitrification both have advantages and disadvantages in terms of cell viability and/or scalability. In the present study, we developed an advanced slow-freezing technique using a programmed freezer with a magnetic field called Cells Alive System (CAS) and examined its effectiveness on human induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NS/PCs). This system significantly increased cell viability after thawing and had less impact on cellular proliferation and differentiation. We further found that frozen-thawed hiPSC-NS/PCs were comparable with non-frozen ones at the transcriptome level. Given these findings, we suggest that the CAS is useful for hiPSC-NS/PCs banking for clinical uses involving neural disorders and may open new avenues for future regenerative medicine.

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.

Evaluation of the immunogenicity of human iPS cell-derived neural stem/progenitor cells in vitro.

To achieve the goal of a first-in-human trial for human induced pluripotent stem cell (hiPSC)-based transplantation for the treatment of various diseases, allogeneic human leukocyte antigen (HLA)-matched hiPSC cell banks represent a realistic tool from the perspective of quality control and cost performance. Furthermore, considering the limited therapeutic time-window for acute injuries, including neurotraumatic injuries, an iPS cell bank is of potential interest. However, due to the relatively immunoprivileged environment of the central nervous system, it is unclear whether HLA matching is required in hiPSC-derived neural stem/progenitor cell (hiPSC-NS/PC) transplantation for the treatment of neurodegenerative diseases and neurotraumatic injuries. In this study, we evaluated the significance of HLA matching in hiPSC-NS/PC transplantation by performing modified mixed lymphocyte reaction (MLR) assays with hiPSC-NS/PCs. Compared to fetus-derived NS/PCs, the expression levels of human leukocyte antigen-antigen D related (HLA-DR) and co-stimulatory molecules on hiPSC-NS/PCs were significantly low, even with the addition of tumor necrosis factor-α (TNFα) and/or interferon-γ (IFNγ) to mimic the inflammatory environment surrounding transplanted hiPSC-NS/PCs in injured tissues. Interestingly, both the allogeneic HLA-matched and the HLA-mismatched responses were similarly low in the modified MLR assay. Furthermore, the autologous response was also similar to the allogeneic response. hiPSC-NS/PCs suppressed the proliferative responses of allogeneic HLA-mismatched peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner. Thus, the low antigen-presenting function and immunosuppressive effects of hiPSC-NS/PCs result in a depressed immune response, even in an allogeneic HLA-mismatched setting. It is crucial to verify whether these in vitro results are reproducible in a clinical setting.