Keiko Sugai

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.

Functional Recovery from Neural Stem/Progenitor Cell Transplantation Combined with Treadmill Training in Mice with Chronic Spinal Cord Injury

Most studies targeting chronic spinal cord injury (SCI) have concluded that neural stem/progenitor cell (NS/PC) transplantation exerts only a subclinical recovery; this in contrast to its remarkable effect on acute and subacute SCI. To determine whether the addition of rehabilitative intervention enhances the effect of NS/PC transplantation for chronic SCI, we used thoracic SCI mouse models to compare manifestations secondary to both transplantation and treadmill training, and the two therapies combined, with a control group. Significant locomotor recovery in comparison with the control group was only achieved in the combined therapy group. Further investigation revealed that NS/PC transplantation improved spinal conductivity and central pattern generator activity, and that treadmill training promoted the appropriate inhibitory motor control. The combined therapy enhanced these independent effects of each single therapy, and facilitated neuronal differentiation of transplanted cells and maturation of central pattern generator activity synergistically. Our data suggest that rehabilitative treatment represents a therapeutic option for locomotor recovery after NS/PC transplantation, even in chronic SCI.

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.

Neural stem/progenitor cell-laden microfibers promote transplant survival in a mouse transected spinal cord injury model

Previous studies have demonstrated that transplantation of neural stem/progenitor cells (NS/PCs) into the lesioned spinal cord can promote functional recovery following incomplete spinal cord injury (SCI) in animal models. However, this strategy is insufficient following complete SCI because of the gap at the lesion epicenter. To obtain functional recovery in a mouse model of complete SCI, this study uses a novel collagen‐based microfiber as a scaffold for engrafted NS/PCs. We hypothesized that the NS/PC–microfiber combination would facilitate lesion closure as well as transplant survival in the transected spinal cord. NS/PCs were seeded inside the novel microfibers, where they maintained their capacity to differentiate and proliferate. After transplantation, the stumps of the transected spinal cord were successfully bridged by the NS/PC‐laden microfibers. Moreover, the transplanted cells migrated into the host spinal cord and differentiated into three neural lineages (astrocytes, neurons, and oligodendrocytes). However, the NS/PC‐laden scaffold could not achieve a neural connection between the rostral end of the injury and the intact caudal area of the spinal cord, nor could it achieve recovery of motor function. To obtain optimal functional recovery, a microfiber design with a modified composition may be useful. Furthermore, combinatorial therapy with rehabilitation and/or medications should also be considered for practical success of biomaterial/cell transplantation‐based approaches to regenerative medicine.

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.

Robust production of human neural cells by establishing neuroepithelial-like stem cells from peripheral blood mononuclear cell-derived feeder-free iPSCs under xeno-free conditions

Neural stem/progenitor cells (NS/PCs) derived from human induced pluripotent stem cells (hiPSCs) are expected to be a valuable cell source for cell therapies that target central nervous system disorders. For clinical applications, NS/PCs should be induced and maintained under clinical grade conditions, which are challenging to achieve. In the present study, we established a procedure to obtain xeno-free long-term self-renewing neuroepithelial-like stem cells (xf-lt-NES cells) from feeder-free hiPSCs using a newly developed xeno-free medium, StemFit(®)AS200. xf-lt-NES cells were cultured for long periods in StemFit(®)AS200 while retaining normal karyotypes, NS/PC marker expression and differentiation capacity for neuronal and glial differentiation in vitro and in vivo. Furthermore, the cells were cryopreserved using a defined serum-free freezing reagent, which demonstrated the feasibility of this xeno-free culture system for large-scale lt-NES cell production and cell banking. Taken together, our system represents a promising approach for the manufacture of clinically relevant products for cell therapy using NS/PCs.

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.