Human-induced pluripotent stem cell-derived neural stem/progenitor cell ex vivo gene therapy with synaptic organizer CPTX for spinal cord injury.

The transplantation of neural stem/progenitor cells (NS/PCs) derived from human induced pluripotent stem cells (hiPSCs) has shown promise in spinal cord injury (SCI) model animals. Establishing a functional synaptic connection between the transplanted and host neurons is crucial for motor function recovery. To boost therapeutic outcomes, we developed an ex vivo gene therapy aimed at promoting synapse formation by expressing the synthetic excitatory synapse organizer CPTX in hiPSC-NS/PCs. Using an immunocompromised transgenic rat model of SCI, we evaluated the effects of transplanting CPTX-expressing hiPSC-NS/PCs using histological and functional analyses. Our findings revealed a significant increase in excitatory synapse formation at the transplantation site. Retrograde monosynaptic tracing indicated extensive integration of transplanted neurons into the surrounding neuronal tracts facilitated by CPTX. Consequently, locomotion and spinal cord conduction significantly improved. Thus, ex vivo gene therapy targeting synapse formation holds promise for future clinical applications and offers potential benefits to individuals with SCI.

Spatial heterogeneity of bone marrow endothelial cells unveils a distinct subtype in the epiphysis.

Bone marrow endothelial cells (BMECs) play a key role in bone formation and haematopoiesis. Although recent studies uncovered the cellular taxonomy of stromal compartments in the bone marrow (BM), the complexity of BMECs is not fully characterized. In the present study, using single-cell RNA sequencing, we defined a spatial heterogeneity of BMECs and identified a capillary subtype, termed type S (secondary ossification) endothelial cells (ECs), exclusively existing in the epiphysis. Type S ECs possessed unique phenotypic characteristics in terms of structure, plasticity and gene expression profiles. Genetic experiments showed that type S ECs atypically contributed to the acquisition of bone strength by secreting type I collagen, the most abundant bone matrix component. Moreover, these cells formed a distinct reservoir for haematopoietic stem cells. These findings provide the landscape for the cellular architecture in the BM vasculature and underscore the importance of epiphyseal ECs during bone and haematopoietic development.

Gene therapy using genome-edited iPS cells for targeting malignant glioma.

Glioblastoma is characterized by diffuse infiltration into the normal brain. Invasive glioma stem cells (GSCs) are an underlying cause of treatment failure. Despite the use of multimodal therapies, the prognosis remains dismal. New therapeutic approach targeting invasive GSCs is required. Here, we show that neural stem cells (NSCs) derived from CRISRP/Cas9-edited human-induced pluripotent stem cell (hiPSC) expressing a suicide gene had higher tumor-trophic migratory capacity compared with mesenchymal stem cells (MSCs), leading to marked in vivo antitumor effects. High migratory capacity in iPSC-NSCs was related to self-repulsive action and pathotropism involved in EphB-ephrinB and CXCL12-CXCR4 signaling. The gene insertion to ACTB provided higher and stable transgene expression than other common insertion sites, such as GAPDH or AAVS1. Ferroptosis was associated with enhanced antitumor immune responses. The thymidylate synthase and dihydroprimidine dehydrogenase expressions predicted the treatment efficacy of therapeutic hiPSC-NSCs. Our results indicate the potential benefit of genome-edited iPS cells based gene therapy for invasive GSCs. Furthermore, the present research concept may become a platform to promote clinical studies using hiPSC.

Mesenchymal properties of iPSC-derived neural progenitors that generate undesired grafts after transplantation.

Although neural stem/progenitor cells derived from human induced pluripotent stem cells (hiPSC-NS/PCs) are expected to be a cell source for cell-based therapy, tumorigenesis of hiPSC-NS/PCs is a potential problem for clinical applications. Therefore, to understand the mechanisms of tumorigenicity in NS/PCs, we clarified the cell populations of NS/PCs. We established single cell-derived NS/PC clones (scNS/PCs) from hiPSC-NS/PCs that generated undesired grafts. Additionally, we performed bioassays on scNS/PCs, which classified cell types within parental hiPSC-NS/PCs. Interestingly, we found unique subsets of scNS/PCs, which exhibited the transcriptome signature of mesenchymal lineages. Furthermore, these scNS/PCs expressed both neural (PSA-NCAM) and mesenchymal (CD73 and CD105) markers, and had an osteogenic differentiation capacity. Notably, eliminating CD73+ CD105+ cells from among parental hiPSC-NS/PCs ensured the quality of hiPSC-NS/PCs. Taken together, the existence of unexpected cell populations among NS/PCs may explain their tumorigenicity leading to potential safety issues of hiPSC-NS/PCs for future regenerative medicine.

Current status and prospects of regenerative medicine for spinal cord injury using human induced pluripotent stem cells: a review.

Spinal cord injury (SCI) is damage to the spinal cord due to trauma or health conditions, resulting in lesions in the spinal cord. Currently, available treatment includes surgical intervention to decompress or stabilize a dislocated loose spine, steroid drugs to reduce inflammation, and subsequent rehabilitation. As there is a rising number of SCI globally, radical treatments to recover spinal cord functions have become highly anticipated. The development of new treatments is indeed progressing. Various therapeutic drug candidates are being developed in clinical trials, including neuroprotective/neurotrophic factors, antibodies for repulsive guidance molecules, and cell transplantation. Among them, with advances in stem cell biology, cell transplantation therapy is currently a promising therapeutic development for SCI. In particular, there have been various reports regarding the realization of regenerative medicine using human induced pluripotent stem cells (iPSCs). This review will introduce the advantages of cell-based therapy based on iPSC-derived neural stem/progenitor cells (iPSC-NS/PCs) and some of their mechanisms of action for functional improvement, which have recently been elucidated. Potential challenges and methodologies to realize the clinical application of iPSC-NS/PCs not only for the subacute phase but also for the chronic phase of SCI will be presented. Finally, we also introduce recent research with a view to the clinical application of spinal cord regenerative therapy and discuss future prospects.

A non-invasive system to monitor in vivo neural graft activity after spinal cord injury.

Expectations for neural stem/progenitor cell (NS/PC) transplantation as a treatment for spinal cord injury (SCI) are increasing. However, whether and how grafted cells are incorporated into the host neural circuit and contribute to motor function recovery remain unknown. The aim of this project was to establish a novel non-invasive in vivo imaging system to visualize the activity of neural grafts by which we can simultaneously demonstrate the circuit-level integration between the graft and host and the contribution of graft neuronal activity to host behaviour. We introduced Akaluc, a newly engineered luciferase, under the control of enhanced synaptic activity-responsive element (E-SARE), a potent neuronal activity-dependent synthetic promoter, into NS/PCs and engrafted the cells into SCI model mice. Through the use of this system, we found that the activity of grafted cells was integrated with host behaviour and driven by host neural circuit inputs. This non-invasive system is expected to help elucidate the therapeutic mechanism of cell transplantation treatment for SCI.

Administration of C5a Receptor Antagonist Improves the Efficacy of Human Induced Pluripotent Stem Cell-Derived Neural Stem/Progenitor Cell Transplantation in the Acute Phase of Spinal Cord Injury.

Human-induced pluripotent stem cell-derived neural stem/progenitor cell (hiPSC-NS/PC) transplantation during the acute phase of spinal cord injury (SCI) is not effective due to the inflammatory response occurring immediately after SCI, which negatively impacts transplanted cell survival. Therefore, we chose to study the powerful chemoattractant complement C5a as a method to generate a more favorable transplantation environment. We hypothesized that suppression of the inflammatory response immediately after SCI by C5a receptor antagonist (C5aRA) would improve the efficacy of hiPSC-NS/PCs transplantation for acute phase SCI. Here, we evaluated the influence of C5aRA on the inflammatory reaction during the acute phase after SCI, and observed significant reductions in several inflammatory cytokines, macrophages, neutrophils, and apoptotic markers. Next, we divided the SCI mice into four groups: 1) phosphate-buffered saline (PBS) only; 2) C5aRA only; 3) PBS + transplantation (PBS+TP); and 4) C5aRA + transplantation (C5aRA+TP). Immediately after SCI, C5aRA or PBS was injected once a day for 4 consecutive days, followed by hiPSC-NS/PC transplantation or PBS into the lesion epicenter on Day 4. The C5aRA+TP group had better functional improvement compared with the PBS only group. The C5aRA+TP group also had a significantly higher cell survival rate compared with the PBS+TP group. This study demonstrates that administration of C5aRA can suppress the inflammatory response during the acute phase of SCI, while improving the survival rate of transplanted hiPSC-NS/PCs, as well as enhancing motor functional restoration. Human-induced pluripotent stem cell-derived neural stem/progenitor cell transplantation with C5aRA is a promising treatment during the acute injury phase for SCI patients.

Modulation by DREADD reveals the therapeutic effect of human iPSC-derived neuronal activity on functional recovery after spinal cord injury.

Transplantation of neural stem/progenitor cells (NS/PCs) derived from human induced pluripotent stem cells (hiPSCs) is considered to be a promising therapy for spinal cord injury (SCI) and will soon be translated to the clinical phase. However, how grafted neuronal activity influences functional recovery has not been fully elucidated. Here, we show the locomotor functional changes caused by inhibiting the neuronal activity of grafted cells using a designer receptor exclusively activated by designer drugs (DREADD). In vitro analyses of inhibitory DREADD (hM4Di)-expressing cells demonstrated the precise inhibition of neuronal activity via administration of clozapine N-oxide. This inhibition led to a significant decrease in locomotor function in SCI mice with cell transplantation, which was exclusively observed following the maturation of grafted neurons. Furthermore, trans-synaptic tracing revealed the integration of graft neurons into the host motor circuitry. These results highlight the significance of engrafting functionally competent neurons by hiPSC-NS/PC transplantation for sufficient recovery from SCI.

FZD5 regulates cellular senescence in human mesenchymal stem/stromal cells.

Human mesenchymal stem/stromal cells (hMSCs) have garnered enormous interest as a potential resource for cell-based therapies. However, the molecular mechanisms regulating senescence in hMSCs remain unclear. To elucidate these mechanisms, we performed gene expression profiling to compare clonal immature MSCs exhibiting multipotency with less potent MSCs. We found that the transcription factor Frizzled 5 (FZD5) is expressed specifically in immature hMSCs. The FZD5 cell surface antigen was also highly expressed in the primary MSC fraction (LNGFR+ THY-1+ ) and cultured MSCs. Treatment of cells with the FZD5 ligand WNT5A promoted their proliferation. Upon FZD5 knockdown, hMSCs exhibited markedly attenuated proliferation and differentiation ability. The observed increase in the levels of senescence markers suggested that FZD5 knockdown promotes cellular senescence by regulating the noncanonical Wnt pathway. Conversely, FZD5 overexpression delayed cell cycle arrest during the continued culture of hMSCs. These results indicated that the intrinsic activation of FZD5 plays an essential role in negatively regulating senescence in hMSCs and suggested that controlling FZD5 signaling offers the potential to regulate hMSC quality and improve the efficacy of cell-replacement therapies using hMSCs.

A robust culture system to generate neural progenitors with gliogenic competence from clinically relevant induced pluripotent stem cells for treatment of spinal cord injury.

Cell-based therapy targeting spinal cord injury (SCI) is an attractive approach to promote functional recovery by replacing damaged tissue. We and other groups have reported the effectiveness of transplanting neural stem/progenitor cells (NS/PCs) derived from human induced pluripotent stem cells (hiPSCs) in SCI animal models for neuronal replacement. Glial replacement is an additional approach for tissue repair; however, the lack of robust procedures to drive iPSCs into NS/PCs which can produce glial cells has hindered the development of glial cell transplantation for the restoration of neuronal functions after SCI. Here, we established a method to generate NS/PCs with gliogenic competence (gNS/PCs) optimized for clinical relevance and utilized them as a source of therapeutic NS/PCs for SCI. We could successfully generate gNS/PCs from clinically relevant hiPSCs, which efficiently produced astrocytes and oligodendrocytes in vitro. We also performed comparison between gNS/PCs and neurogenic NS/PCs based on single cell RNA-seq analysis and found that gNS/PCs were distinguished by expression of several transcription factors including HEY2 and NFIB. After gNS/PC transplantation, the graft did not exhibit tumor-like tissue formation, indicating the safety of them as a source of cell therapy. Importantly, the gNS/PCs triggered functional recovery in an SCI animal model, with remyelination of demyelinated axons and improved motor function. Given the inherent safety of gNS/PCs and favorable outcomes observed after their transplantation, cell-based medicine using the gNS/PCs-induction procedure described here together with clinically relevant iPSCs is realistic and would be beneficial for SCI patients.

The adeno-associated virus rh10 vector is an effective gene transfer system for chronic spinal cord injury.

Treatment options for chronic spinal cord injury (SCI) remain limited due to unfavourable changes in the microenvironment. Gene therapy can overcome these barriers through continuous delivery of therapeutic gene products to the target tissue. In particular, adeno-associated virus (AAV) vectors are potential candidates for use in chronic SCI, considering their safety and stable gene expression in vivo. Given that different AAV serotypes display different cellular tropisms, it is extremely important to select an optimal serotype for establishing a gene transfer system during the chronic phase of SCI. Therefore, we generated multiple AAV serotypes expressing ffLuc-cp156, a fusion protein of firefly luciferase and Venus, a variant of yellow fluorescent protein with fast and efficient maturation, as a reporter, and we performed intraparenchymal injection in a chronic SCI mouse model. Among the various serotypes tested, AAVrh10 displayed the highest photon count on bioluminescence imaging. Immunohistological analysis revealed that AAVrh10 showed favourable tropism for neurons, astrocytes, and oligodendrocytes. Additionally, with AAVrh10, the area expressing Venus was larger in the injury epicentre and extended to the surrounding tissue. Furthermore, the fluorescence intensity was significantly higher with AAVrh10 than with the other vectors. These results indicate that AAVrh10 may be an appropriate serotype for gene delivery to the chronically injured spinal cord. This promising tool may be applied for research and development related to the treatment of chronic SCI.

Loss of TET proteins in regulatory T cells promotes abnormal proliferation, Foxp3 destabilization and IL-17 expression.

Ten-eleven translocation (TET) proteins regulate DNA methylation and gene expression by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Although Tet2/Tet3 deficiency has been reported to lead to myeloid cell, B-cell and invariant natural killer T (iNKT) cell malignancy, the effect of TET on regulatory T cells (Tregs) has not been elucidated. We found that Tet2/Tet3 deficiency in Tregs led to lethal hyperproliferation of CD4+Foxp3+ T cells in the spleen and mesenteric lymph nodes after 5 months of age. Additionally, in aged Treg-specific Tet2/Tet3-deficient mice, serum IgG1, IgG3, IgM and IgE levels were markedly elevated. High IL-17 expression was observed in both Foxp3+ and Fopx3- CD4+ T cells, and adoptive transfer of Tet2/Tet3-deficient Tregs into lymphopenic mice inhibited Foxp3 expression and caused conversion into IL-17-producing cells. However, the conserved non-coding DNA sequence-2 (CNS2) region of the Foxp3 gene locus, which has been shown to be particularly important for stable Foxp3 expression, was only partly methylated. We identified novel TET-dependent demethylation sites in the Foxp3 upstream enhancer, which may contribute to stable Foxp3 expression. Together, these data indicate that Tet2 and Tet3 are involved in Treg stability and immune homeostasis in mice.

Selective Ablation of Tumorigenic Cells Following Human Induced Pluripotent Stem Cell-Derived Neural Stem/Progenitor Cell Transplantation in Spinal Cord Injury.

Tumorigenesis is an important problem that needs to be addressed in the field of human stem/progenitor cell transplantation for the treatment of subacute spinal cord injury (SCI). When certain "tumorigenic" cell lines are transplanted into the spinal cord of SCI mice model, there is initial improvement of motor function, followed by abrupt deterioration secondary to the effect of tumor growth. A significant proportion of the transplanted cells remains undifferentiated after transplantation and is thought to increase the risk of tumorigenesis. In this study, using lentiviral vectors, we introduced the herpes simplex virus type 1 thymidine kinase (HSVtk) gene into a human induced pluripotent stem cell-derived neural stem/progenitor cell (hiPSC-NS/PC) line that is known to undergo tumorigenic transformation. Such approach enables selective ablation of the immature proliferating cells and thereby prevents subsequent tumor formation. In vitro, the HSVtk system successfully ablated the immature proliferative neural cells while preserving mature postmitotic neuronal cells. Similar results were observed in vivo following transplantation into the injured spinal cords of immune-deficient (nonobese diabetic-severe combined immune-deficient) mice. Ablation of the proliferating cells exerted a protective effect on the motor function which was regained after transplantation, simultaneously defending the spinal cord from the harmful tumor growth. These results suggest a potentially promising role of suicide genes in opposing tumorigenesis during stem cell therapy. This system allows both preventing and treating tumorigenesis following hiPSC-NS/PC transplantation without sacrificing the improved motor function. Stem Cells Translational Medicine 2019;8:260&270.

Concise Review: Laying the Groundwork for a First-In-Human Study of an Induced Pluripotent Stem Cell-Based Intervention for Spinal Cord Injury.

There have been numerous attempts to develop stem cell transplantation approaches to promote the regeneration of spinal cord injury (SCI). Our multicenter team is currently planning to launch a first-in-human clinical study of an induced pluripotent stem cell (iPSC)-based cell transplant intervention for subacute SCI. This trial was conducted as class I regenerative medicine protocol as provided for under Japan's Act on the Safety of Regenerative Medicine, using neural stem/progenitor cells derived from a clinical-grade, integration-free human "iPSC stock" generated by the Kyoto University Center for iPS Cell Research and Application. In the present article, we describe how we are preparing to initiate this clinical study, including addressing the issues of safety and tumorigenesis as well as practical problems that must be overcome to enable the development of therapeutic interventions for patients with chronic SCI. Stem Cells 2019;37:6-13.

Treatment with a Gamma-Secretase Inhibitor Promotes Functional Recovery in Human iPSC- Derived Transplants for Chronic Spinal Cord Injury.

Treatment involving regenerative medicine for chronic spinal cord injury (SCI) is difficult due to phase-dependent changes in the intraspinal environment. We previously reported that treatment with a gamma-secretase inhibitor (GSI), which inhibits Notch signaling, promotes the differentiation into mature neurons in human induced pluripotent stem cell-derived neural stem/progenitor cell (hiPSC-NS/PC) transplantation for subacute SCI. Here, we evaluated the efficacy of GSI-treated hiPSC-NS/PC transplantation in treating chronic SCI, which resulted in significantly enhanced axonal regrowth, remyelination, inhibitory synapse formation with the host neural circuitry, and reticulo spinal tract fiber formation. Interestingly, inhibiting Notch signaling with GSI caused phosphorylation of p38 MAPK, which is a key molecule required to promote axonal regeneration. These favorable outcomes contributed to motor function improvement. Therefore, treating cells with GSI provides a beneficial effect after transplantation, even in the chronic phase following SCI.

CHARGE syndrome modeling using patient-iPSCs reveals defective migration of neural crest cells harboring CHD7 mutations.

CHARGE syndrome is caused by heterozygous mutations in the chromatin remodeler, CHD7, and is characterized by a set of malformations that, on clinical grounds, were historically postulated to arise from defects in neural crest formation during embryogenesis. To better delineate neural crest defects in CHARGE syndrome, we generated induced pluripotent stem cells (iPSCs) from two patients with typical syndrome manifestations, and characterized neural crest cells differentiated in vitro from these iPSCs (iPSC-NCCs). We found that expression of genes associated with cell migration was altered in CHARGE iPSC-NCCs compared to control iPSC-NCCs. Consistently, CHARGE iPSC-NCCs showed defective delamination, migration and motility in vitro, and their transplantation in ovo revealed overall defective migratory activity in the chick embryo. These results support the historical inference that CHARGE syndrome patients exhibit defects in neural crest migration, and provide the first successful application of patient-derived iPSCs in modeling craniofacial disorders.

Low immunogenicity of mouse induced pluripotent stem cell-derived neural stem/progenitor cells.

Resolving the immunogenicity of cells derived from induced pluripotent stem cells (iPSCs) remains an important challenge for cell transplant strategies that use banked allogeneic cells. Thus, we evaluated the immunogenicity of mouse fetal neural stem/progenitor cells (fetus-NSPCs) and iPSC-derived neural stem/progenitor cells (iPSC-NSPCs) both in vitro and in vivo. Flow cytometry revealed the low expression of immunological surface antigens, and these cells survived in all mice when transplanted syngeneically into subcutaneous tissue and the spinal cord. In contrast, an allogeneic transplantation into subcutaneous tissue was rejected in all mice, and allogeneic cells transplanted into intact and injured spinal cords survived for 3 months in approximately 20% of mice. In addition, cell survival was increased after co-treatment with an immunosuppressive agent. Thus, the immunogenicity and post-transplantation immunological dynamics of iPSC-NSPCs resemble those of fetus-NSPCs.

Improvement of Foxp3 stability through CNS2 demethylation by TET enzyme induction and activation.

Since induced regulatory T cells (iTregs) can be produced in a large quantity in vitro, these cells are expected to be clinically useful to induce immunological tolerance in various immunological diseases. Foxp3 (Forkhead box P3) expression in iTregs is, however, unstable due to the lack of demethylation of the CpG island in the conserved non-coding sequence 2 (CNS2) of the Foxp3 locus. To facilitate the demethylation of CNS2, we over-expressed the catalytic domain (CD) of the ten-eleven translocation (TET) protein, which catalyzes the steps of the iterative demethylation of 5-methylcytosine. TET-CD over-expression in iTregs resulted in partial demethylation of CNS2 and stable Foxp3 expression. We also discovered that TET expression was enhanced under low oxygen (5%) culture conditions, which facilitated CNS2 DNA demethylation and stabilization of Foxp3 expression in a TET2- and TET3-dependent manner. In combination with vitamin C treatment, which has been reported to enhance TET catalytic activity, iTregs generated under low oxygen conditions retained more stable Foxp3 expression in vitro and in vivo and exhibited stronger suppression activity in a colitis model compared with untreated iTregs. Our data indicate that the induction and activation of TET enzymes in iTregs would be an effective method for Treg-mediated adoptive immunotherapy.

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

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.

Whole-Genome DNA Methylation Analyses Revealed Epigenetic Instability in Tumorigenic Human iPS Cell-Derived Neural Stem/Progenitor Cells.

Although human induced pluripotent stem cell (hiPSC) derivatives are considered promising cellular resources for regenerative medicine, their tumorigenicity potentially limits their clinical application in hiPSC technologies. We previously demonstrated that oncogenic hiPSC-derived neural stem/progenitor cells (hiPSC-NS/PCs) produced tumor-like tissues that were distinct from teratomas. To gain insight into the mechanisms underlying the regulation of tumorigenicity in hiPSC-NS/PCs, we performed an integrated analysis using the Infinium HumanMethylation450 BeadChip array and the HumanHT-12 v4.0 Expression BeadChip array to compare the comprehensive DNA methylation and gene expression profiles of tumorigenic hiPSC-NS/PCs (253G1-NS/PCs) and non-tumorigenic cells (201B7-NS/PCs). Although the DNA methylation profiles of 253G1-hiPSCs and 201B7-hiPSCs were similar regardless of passage number, the methylation status of the global DNA methylation profiles of 253G1-NS/PCs and 201B7-NS/PCs differed; the genomic regions surrounding the transcriptional start site of the CAT and PSMD5 genes were hypermethylated in 253G1-NS/PCs but not in 201B7-NS/PCs. Interestingly, the aberrant DNA methylation profile was more pronounced in 253G1-NS/PCs that had been passaged more than 15 times. In addition, we identified aberrations in DNA methylation at the RBP1 gene locus; the DNA methylation frequency in RBP1 changed as 253G1-NS/PCs were sequentially passaged. These results indicate that different NS/PC clones have different DNA methylomes and that DNA methylation patterns are unstable as cells are passaged. Therefore, DNA methylation profiles should be included in the criteria used to evaluate the tumorigenicity of hiPSC-NS/PCs in the clinical setting. Stem Cells 2017;35:1316-1327.