The past decade has witnessed an extremely rapid increase in the number of newly established stem cell lines. However, due to the lack of a standardized format, data exchange among stem cell line resources has been challenging, and no system can search all stem cell lines across resources worldwide. To solve this problem, we have developed the Integrated Collection of Stem Cell Bank data (ICSCB) (http://icscb.stemcellinformatics.org/), the largest database search portal for stem cell line information, based on the standardized data items and terms of the MIACARM framework. Currently, ICSCB can retrieve >16,000 cell lines from four major data resources in Europe, Japan, and the United States. ICSCB is automatically updated to provide the latest cell line information, and its integrative search helps users collect cell line information for over 1,000 diseases, including many rare diseases worldwide, which has been a formidable task, thereby distinguishing itself from other database search portals.

Amyotrophic lateral sclerosis (ALS) is a late-onset motor neuron disorder. Although its neuropathology is well understood, the cellular and molecular mechanisms are yet to be elucidated due to limitations in the currently available human genetic data. In this study, we generated induced pluripotent stem cells (iPSC) from two familial ALS (FALS) patients with a missense mutation in the fused-in sarcoma (FUS) gene carrying the heterozygous FUS H517D mutation, and isogenic iPSCs with the homozygous FUS H517D mutation by genome editing technology. These cell-derived motor neurons mimicked several neurodegenerative phenotypes including mis-localization of FUS into cytosolic and stress granules under stress conditions, and cellular vulnerability. Moreover, exon array analysis using motor neuron precursor cells (MPCs) combined with CLIP-seq datasets revealed aberrant gene expression and/or splicing pattern in FALS MPCs. These results suggest that iPSC-derived motor neurons are a useful tool for analyzing the pathogenesis of human motor neuron disorders.

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.

Amyotrophic lateral sclerosis (ALS) is an adult-onset incurable motor neuron (MN) disease. The reasons for selective MN vulnerability in ALS are unknown. Axonal pathology is among the earliest signs of ALS. We searched for novel modulatory genes in human MN axon shortening affected by TARDBP mutations. In transcriptome analysis of RNA present in the axon compartment of human-derived induced pluripotent stem cell (iPSC)-derived MNs, PHOX2B (paired-like homeobox protein 2B) showed lower expression in TARDBP mutant axons, which was consistent with axon qPCR and in situ hybridization. PHOX2B mRNA stability was reduced in TARDBP mutant MNs. Furthermore, PHOX2B knockdown reduced neurite length in human MNs. Finally, phox2b knockdown in zebrafish induced short spinal axons and impaired escape response. PHOX2B is known to be highly express in other types of neurons maintained after ALS progression. Collectively, TARDBP mutations induced loss of axonal resilience, which is an important ALS-related phenotype mediated by PHOX2B downregulation.

Previously, we described the safety and therapeutic potential of neurospheres (NSs) derived from a human induced pluripotent stem cell (iPSC) clone, 201B7, in a spinal cord injury (SCI) mouse model. However, several safety issues concerning iPSC-based cell therapy remain unresolved. Here, we investigated another iPSC clone, 253G1, that we established by transducing OCT4, SOX2, and KLF4 into adult human dermal fibroblasts collected from the same donor who provided the 201B7 clone. The grafted 253G1-NSs survived, differentiated into three neural lineages, and promoted functional recovery accompanied by stimulated synapse formation 47 days after transplantation. However, long-term observation (for up to 103 days) revealed deteriorated motor function accompanied by tumor formation. The tumors consisted of Nestin(+) undifferentiated neural cells and exhibited activation of the OCT4 transgene. Transcriptome analysis revealed that a heightened mesenchymal transition may have contributed to the progression of tumors derived from grafted cells.

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.

Parkinson disease (PD) is a progressive neurological disease caused by selective degeneration of dopaminergic (DA) neurons in the substantia nigra. Although most cases of PD are sporadic cases, familial PD provides a versatile research model for basic mechanistic insights into the pathogenesis of PD. In this study, we generated DA neurons from PARK2 patient-specific, isogenic PARK2 null and PARK6 patient-specific induced pluripotent stem cells and found that these neurons exhibited more apoptosis and greater susceptibility to rotenone-induced mitochondrial stress. From phenotypic screening with an FDA-approved drug library, one voltage-gated calcium channel antagonist, benidipine, was found to suppress rotenone-induced apoptosis. Furthermore, we demonstrated the dysregulation of calcium homeostasis and increased susceptibility to rotenone-induced stress in PD, which is prevented by T-type calcium channel knockdown or antagonists. These findings suggest that calcium homeostasis in DA neurons might be a useful target for developing new drugs for PD patients.

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.

Several groups have reported the existence of a form of pluripotency that resembles that of mouse embryonic stem cells (mESCs), i.e., a naive state, in human pluripotent stem cells; however, the characteristics vary between reports. The nuclear receptor ESRRB is expressed in mESCs and plays a significant role in their self-renewal, but its expression has not been observed in most naive-like human induced pluripotent stem cells (hiPSCs). In this study, we modified several methods for converting hiPSCs into a naive state through the transgenic expression of several reprogramming factors. The resulting cells express the components of the core transcriptional network of mESCs, including ESRRB, at high levels, which suggests the existence of naive-state hiPSCs that are similar to mESCs. We also demonstrate that these cells differentiate more readily into neural cells than do conventional hiPSCs. These features may be beneficial for their use in disease modeling and regenerative medicine.

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.

Human neural precursor cells (hNPCs) derived from pluripotent stem cells display a high propensity for neuronal differentiation, but they require long-term culturing to differentiate efficiently into astrocytes. The mechanisms underlying this biased fate specification of hNPCs remain elusive. Here, we show that hypoxia confers astrocytic differentiation potential on hNPCs through epigenetic gene regulation, and that this was achieved by cooperation between hypoxia-inducible factor 1α and Notch signaling, accompanied by a reduction of DNA methylation level in the promoter region of a typical astrocyte-specific gene, Glial fibrillary acidic protein. Furthermore, we found that this hypoxic culture condition could be applied to rapid generation of astrocytes from Rett syndrome patient-derived hNPCs, and that these astrocytes impaired neuronal development. Thus, our findings shed further light on the molecular mechanisms regulating hNPC differentiation and provide attractive tools for the development of therapeutic strategies for treating astrocyte-mediated neurological disorders.

Although pluripotent stem cells can generate various types of differentiated cells, it is unclear why lineage-committed stem/progenitor cells derived from pluripotent stem cells are decelerated and why the differentiation-resistant propensity of embryonic stem cell (ESC)/induced pluripotent stem cell (iPSC)-derived cells is predominant compared with the in vivo equivalents derived from embryonic/adult tissues. In this study, we demonstrated that iPSCs reprogrammed and maintained with three chemical inhibitors of the fibroblast growth factor 4-mitogen-activated protein kinase cascade and GSK3β (3i) could be differentiated into all three germ layers more efficiently than the iPSCs reprogrammed without the 3i chemicals, even though they were maintained with 3i chemicals once they were reprogrammed. Although the iPSCs reprogrammed with 3i had increased numbers of Zscan4-positive cells, the Zscan4-positive cells among iPSCs that were reprogrammed without 3i did not have an accelerated differentiation ability. These observations suggest that 3i exposure during the reprogramming period determines the accelerated differentiation/maturation potentials of iPSCs that are stably maintained at the distinct state.

The CNS contains many diverse neuronal subtypes, and most neurological diseases target specific subtypes. However, the mechanism of neuronal subtype specificity of disease phenotypes remains elusive. Although in vitro disease models employing human pluripotent stem cells (PSCs) have great potential to clarify the association of neuronal subtypes with disease, it is currently difficult to compare various PSC-derived subtypes. This is due to the limited number of subtypes whose induction is established, and different cultivation protocols for each subtype. Here, we report a culture system to control the regional identity of PSC-derived neurons along the anteroposterior (A-P) and dorsoventral (D-V) axes. This system was successfully used to obtain various neuronal subtypes based on the same protocol. Furthermore, we reproduced subtype-specific phenotypes of amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD) by comparing the obtained subtypes. Therefore, our culture system provides new opportunities for modeling neurological diseases with PSCs.

Human pluripotent stem cells (hPSCs) represent a potentially valuable cell source for applications in cell replacement therapy, drug development, and disease modeling. For all these uses, it is necessary to develop reproducible and robust protocols for differentiation into desired cell types. However, differentiation protocols remain unstable and inefficient, which makes minimizing the differentiation variance among hPSC lines and obtaining purified terminally differentiated cells extremely time consuming. Here, we report a simple treatment with three small molecules-SB431542, dorsomorphine, and CHIR99021-that enhanced hPSC differentiation into three germ layers with a chemically transitional embryoid-body-like state (CTraS). Induction of CTraS reduced the innate differentiation propensities of hPSCs (even unfavorably differentiated hPSCs) and shifted their differentiation into terminally differentiated cells, particularly neurons. In addition, CTraS induction accelerated in vitro pathological expression concurrently with neural maturation. Thus, CTraS can promote the latent potential of hPSCs for differentiation and potentially expand the utility and applicability of hPSCs.

Mutations in the microtubule-associated protein tau (MAPT) gene are known to cause familial frontotemporal dementia (FTD). The R406W tau mutation is a unique missense mutation whose patients have been reported to exhibit Alzheimer's disease (AD)-like phenotypes rather than the more typical FTD phenotypes. In this study, we established patient-derived induced pluripotent stem cell (iPSC) models to investigate the disease pathology induced by the R406W mutation. We generated iPSCs from patients and established isogenic lines using CRISPR/Cas9. The iPSCs were induced into cerebral organoids, which were dissociated into cortical neurons with high purity. In this neuronal culture, the mutant tau protein exhibited reduced phosphorylation levels and was increasingly fragmented by calpain. Furthermore, the mutant tau protein was mislocalized and the axons of the patient-derived neurons displayed morphological and functional abnormalities, which were rescued by microtubule stabilization. The findings of our study provide mechanistic insight into tau pathology and a potential for therapeutic intervention.

The concept of a perivascular niche has been proposed for neural stem cells (NSCs). This study examined endothelial colony-forming cell (ECFC)-secreted proteins as potential niche factors for NSCs. Intraventricle infusion with ECFC-secreted proteins increased the number of NSCs. ECFC-secreted proteins were more effective in promoting NSC self-renewal than marrow stromal cell (MSC)-secreted proteins. Differential proteomics analysis of MSC-secreted and ECFC-secreted proteins was performed, which revealed chitinase-like protein 3 (CHIL3; also called ECF-L or Ym1) as a candidate niche factor for NSCs. Experiments with recombinant CHIL3, small interfering RNA, and neutralizing antibodies demonstrated that CHIL3 stimulated NSC self-renewal with neurogenic propensity. CHIL3 was endogenously expressed in the neurogenic niche of the brain and retina as well as in the injured brain and retina. Transcriptome and phosphoproteome analyses revealed that CHIL3 activated various genes and proteins associated with NSC maintenance or neurogenesis. Thus, CHIL3 is a novel niche factor for NSCs.

Functional recovery is still limited mainly due to several mechanisms, such as the activation of Nogo receptor-1 (NgR1) signaling, when human induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NS/PC) are transplanted for subacute spinal cord injury (SCI). We previously reported the neuroprotective and regenerative benefits of overexpression of lateral olfactory tract usher substance (LOTUS), an endogenous NgR1 antagonist, in the injured spinal cord using transgenic mice. Here, we evaluate the effects of lentiviral transduction of LOTUS gene into hiPSC-NS/PCs before transplantation in a mouse model of subacute SCI. The transduced LOTUS contributes to neurite extension, suppression of apoptosis, and secretion of neurotrophic factors in vitro. In vivo, the hiPSC-NS/PCs enhance the survival of grafted cells and enhance axonal extension of the transplanted cells, resulting in significant restoration of motor function following SCI. Therefore, the gene transduction of LOTUS in hiPSC-NS/PCs could be a promising adjunct for transplantation therapy for SCI.

Neural stem/progenitor cell (NSPC) transplantation is a promising therapeutic strategy for spinal cord injury (SCI). However, the efficacy of NSPC transplantation on severe SCI is poorly understood. We herein show that NSPC transplantation promotes functional recovery after mild and moderate SCI, but not after severe SCI. In severe SCI mice, there were few remaining host neurons within the range of NSPC engraftment; thus, we examined whether the co-distribution of transplant and host is a contributory factor for functional improvement. A cellular selective analysis using laser microdissection revealed that drug-induced host neuronal ablation considerably decreased the synaptogenic potential of the engrafted NSPCs. Furthermore, following host neuronal ablation, neuronal retrograde tracing showed less propriospinal relay connections bridging the lesion after NSPC transplantation. Our findings suggest that the interactive synaptic reorganization between engrafted NSPCs and spared host neurons is crucial for functional recovery, providing significant insight for establishing therapeutic strategies for severe SCI.

Neurogenesis in specific brain regions in adult mammals decreases with age. Progressive reduction in the proliferation of neural stem and progenitor cells (NS/PCs) is a primary cause of this age-associated decline. However, the mechanism responsible for this reduction is poorly understood. We identify p38 MAPK as a key factor in the proliferation of neural progenitor cells (NPCs) in adult neurogenic niches. p38 expression in adult NS/PCs is downregulated during aging. Deletion of p38α in NS/PCs specifically reduces the proliferation of NPCs but not stem cells. Conversely, forced expression of p38α in NS/PCs in the aged mouse subventricular zone (SVZ) restores NPC proliferation and neurogenesis, and prevents age-dependent SVZ atrophy. We also found that p38 is necessary for suppressing the expression of Wnt antagonists DKK1 and SFRP3, which inhibit the proliferation of NPCs. Age-related reduction in p38 thus leads to decreased adult neurogenesis via downregulation of Wnt signaling.

The emergence of single-cell RNA sequencing (RNA-seq) has radically changed the observation of cellular diversity. Although annotations of RNA-seq data require preserved properties among cells of an identity, annotations using conventional methods have not been able to capture universal characters of a cell type. Analysis of expression levels cannot be accurately annotated for cells because differences in transcription do not necessarily explain biological characteristics in terms of cellular functions and because the data themselves do not inform about the correct mapping between cell types and genes. Hence, in this study, we developed a new representation of cellular identities that can be compared over different datasets while preserving nontrivial biological semantics. To generalize the notion of cell types, we developed a new framework to manage cellular identities in terms of set theory. We provided further insights into cells by installing mathematical descriptions of cell biology. We also performed experiments that could correspond to practical applications in annotations of RNA-seq data.