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.

Chromatin remodeler CHD7 targets active enhancer region to regulate cell type-specific gene expression in human neural crest cells.

A mutation in the chromatin remodeler chromodomain helicase DNA-binding 7 (CHD7) gene causes the multiple congenital anomaly CHARGE syndrome. The craniofacial anomalies observed in CHARGE syndrome are caused by dysfunctions of neural crest cells (NCCs), which originate from the neural tube. However, the mechanism by which CHD7 regulates the function of human NCCs (hNCCs) remains unclear. We aimed to characterize the cis-regulatory elements governed by CHD7 in hNCCs by analyzing genome-wide ChIP-Seq data and identifying hNCC-specific CHD7-binding profiles. We compared CHD7-binding regions among cell types, including human induced pluripotent stem cells and human neuroepithelial cells, to determine the comprehensive properties of CHD7-binding in hNCCs. Importantly, analysis of the hNCC-specific CHD7-bound region revealed transcription factor AP-2α as a potential co-factor facilitating the cell type-specific transcriptional program in hNCCs. CHD7 was strongly associated with active enhancer regions, permitting the expression of hNCC-specific genes to sustain the function of hNCCs. Our findings reveal the regulatory mechanisms of CHD7 in hNCCs, thus providing additional information regarding the transcriptional programs in hNCCs.

Human PSCs determine the competency of cerebral organoid differentiation via FGF signaling and epigenetic mechanisms.

Various culture methods have been developed for maintaining human pluripotent stem cells (PSCs). These PSC maintenance methods exhibit biased differentiation; for example, feeder-dependent PSCs efficiently yield cerebral organoids, but it is difficult to generate organoids from feeder-free PSCs. It remains unknown how PSC maintenance conditions affect differentiation. In this study, we identified fibroblast growth factor (FGF) signaling in feeder-free PSC maintenance as a key factor that determines the differentiation toward cerebral organoids. The inhibition of FGF signaling in feeder-free PSCs rescued organoid generation to the same level in feeder-dependent cultures. FGF inhibition induced DNA methylation at the WNT5A locus, and this epigenetic change suppressed the future activation of non-canonical Wnt signaling after differentiation, leading to reliable cerebral organoid generation. This study underscores the importance of PSC culture conditions for directed differentiation into cerebral organoids, and the epigenetic status regulated by FGF signaling is involved in the underlying mechanisms.

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.

Coupling of angiogenesis and odontogenesis orchestrates tooth mineralization in mice.

The skeletal system consists of bones and teeth, both of which are hardened via mineralization to support daily physical activity and mastication. The precise mechanism for this process, especially how blood vessels contribute to tissue mineralization, remains incompletely understood. Here, we established an imaging technique to visualize the 3D structure of the tooth vasculature at a single-cell level. Using this technique combined with single-cell RNA sequencing, we identified a unique endothelial subtype specialized to dentinogenesis, a process of tooth mineralization, termed periodontal tip-like endothelial cells. These capillaries exhibit high angiogenic activity and plasticity under the control of odontoblasts; in turn, the capillaries trigger odontoblast maturation. Metabolomic analysis demonstrated that the capillaries perform the phosphate delivery required for dentinogenesis. Taken together, our data identified the fundamental cell-to-cell communications that orchestrate tooth formation, angiogenic-odontogenic coupling, a distinct mechanism compared to the angiogenic-osteogenic coupling in bones. This mechanism contributes to our understanding concerning the functional diversity of organotypic vasculature.

Caregiver perceptions of sleep problems and desired areas of change in young children.

OBJECTIVE: To explore the prevalence of and relationship between caregiver-reported sleep problems and sleep-related desired areas of change in young children (0-36 months) in a multinational sample. METHODS: Caregivers (96.5% mothers) of 2219 young children (birth to 3 years; M = 13.7 mos; 49.8% male) completed an online survey including an abbreviated Brief Infant Sleep Questionnaire-Revised (BISQ-R) and questions about desired areas of change regarding their child's sleep. Data were collected in six countries (Indonesia, Japan, New Zealand, Singapore, Thailand, and United States). RESULTS: Overall, 35% of caregivers reported a sleep problem and nearly all (96%) indicated a desired area of change, with 76% endorsing changes in 3 or more categories (bedtime/falling asleep, overnight, morning, and naps). Desiring a change in their child's sleep was universal across age group and country, with those perceiving a sleep problem more likely to endorse an area of change than those without a sleep problem. Overall, the top change categories were bedtime (80%), naps (74%), and overnight (67%). Top specific areas of change related to sleeping for longer stretches, waking up later in the morning, and having an earlier bedtime. CONCLUSIONS: Although one-third of caregivers perceived that their child had a sleep problem, nearly all caregivers identified desired areas of change related to their child's sleep, across the first three years of life and all countries. Sleep education, such as normalizing sleep challenges that are developmentally appropriate, is warranted for all families of young children, regardless of whether sleep problems are endorsed.

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.

Which Is More Important for Health: Sleep Quantity or Sleep Quality?

Sleep is one of the basic physiological processes for human survival. Both sleep quantity and sleep quality are fundamental components of sleep. This review looks at both sleep quantity and sleep quality, considering how to manage the complex but probably unavoidable physiological phenomenon of sleep. The need for sleep has marked variations between individuals, in addition to the effects of variable conditions. Studies on sleep quality started later than those on sleep quantity, beginning in 1989 when Ford and Kamerow revealed that insomnia increases the risk of psychiatric disorders. According to the nationwide research team on the quality of sleep (19FA0901), sleep quality is superior to sleep quantity as an index for assessing sleep, and that restfulness obtained through sleep is a useful index for assessing sleep quality. We should pay more attention to obtaining sleep of good quality (restfulness, no sleepiness, no need for more sleep, sufficient objective sleep depth, etc.), although there have not been enough studies on the associations between sleep quality and health or disorders in children and adolescents. Further studies using the deviation from an individual's optimal sleep quantity may show us another aspect of the effects of sleep quantity on various life issues.

Factors Affecting the Quality of Sleep in Children.

Sleep quality is difficult to define objectively [...].

Features of Pupils in Each Dinner Habit: Non-late Regular, Regular but Late, and Irregular.

BACKGROUND: Both late and irregular caloric intakes are becoming common, especially among young adults. This study aimed to investigate features of pupils in Japan following different dinner habits. METHODS: Dinner habits were categorized into 3 types: non-late regular, regular but late, and irregular. Questionnaires of 2722 pupils in grades 5 to 12 were included, and a multiple comparison test was conducted to investigate the differences of lifestyle factors among the 3 dinnerpattern categories. RESULTS: Only half of grade 5 and 6 pupils had non-late regular dinners. The rate of pupils who had regular but late dinners increased from elementary school (11.8%) to senior high school (41.1%) via junior high school (15.1%). More than 30% of pupils took dinner irregularly. The pupils taking non-late regular dinner showed the earliest bedtime and the longest sleep duration among the 3 dinner-time habit categories and also revealed the best academic performance and sleepiness scores. The pupils taking regular but late dinner showed the highest grade, the latest bedtime, the shortest sleep duration, the longest school-day screen time scores, and the worst sleepiness and self-reported academic performance scores. Irregular dinner-takers showed the longest after-school activities. CONCLUSIONS: Further attention should be given to both late and irregular dinner habits of pupils in terms of supporting their health. Further studies are needed to recommend suitable dinner timings for pupils by assessing optimal levels of variable lifestyle factors, including after-school activity.

Lifestyle habits associated with screen time among pupils in Japan.

BACKGROUND: Media use is pervasive among pupils. This study aimed to determine lifestyle factors associated with screen time. METHODS: The study used a cross-sectional design, and 2,722 questionnaires obtained from pupils in grades 5-12 were analyzed. Multiple regression analysis was used to determine significant lifestyle factors associated with screen time. Grade, gender, bedtime and waking time on both school days and non-school days, academic performance, sleepiness, breakfast, dinner regularity, defecation habits, hours of after-school activities, physical activity, and body mass index were used as the variables. RESULTS: Significant regression formulae were obtained for all school types: adjusted R2 /P values were 0.21/<0.001 for elementary school, 0.21/<0.001 for junior high school, and 0.14/<0.001 for high school. Later non-school-day bedtime (standardized regression coefficient/P values were 0.14/< 0.001 for elementary school, 0.14/<0.001 for junior high school, and 0.09/<0.05 for high school) was significantly associated with increased screen time for all school types. In both elementary and junior high schools, more sleepiness (0.12/<0.001 for elementary school, 0.13/<0.001 for junior high school), shorter after-school activity (-0.24/<0.001 for elementary school, -0.19/<0.001 for junior high school), and higher standardized body mass index (0.08/<0.05 for elementary school, 0.08/<0.01 for junior high school) were significantly associated with screen time increase. In both junior and senior high schools, breakfast skipping (0.15/<0.001 for junior school, 0.14/<0.001 for high school) revealed a significant association with screen time increase. CONCLUSIONS: Media use is associated with variable lifestyle habits. Effective approaches to reduce heavy media use remain to be determined.

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.

Pupils with negative social jetlag in Japan are hypothesised to constitute a discrete population.

Social jetlag (SJL) is the difference between the midpoint time of sleep between weekend and weekday. SJL is experienced by many adolescents and is a matter of concern in public health. In private experiences, pupils who were forced to wake up early on non-school day mornings to engage in hard physical activity and who had little time for sleep on non-school nights were identified. These pupils are supposed to show negative SJL values. However, few studies have described pupils with negative SJL values. The current study aimed to raise a hypothesis that pupils with negative SJL might constitute a discrete population with typical characteristic features. A total of 2,722 questionnaires obtained from pupils in grades 5 to 12 were assessed by the multiple comparison test of the Dunnett method to determine significant differences in the lifestyle habits among SJL categories (SJL-I, <0 h; SJL-II, 0-1 h; SJL-III, 1-2 h; SJL-IV, 2 h or more). Pupils with SJL-I were mostly male compared to those in SJL-II. Physical activity score showed a significantly higher value in SJL-I than SJL-II, while that in SJL-III and SJL-IV revealed significantly lower values than that in SJL-II. Contrarily, non-school day screen time and non-school night sleep duration showed significantly lower values in SJL-I than SJL-II, while those in SJL-III and SJL-IV revealed significantly higher values than those in SJL-II. After-school activity score showed a significantly higher value in SJL-I than SJL-II, while that in SJL-III and SJL-IV revealed lower values than that in SJL-II. At least in Japan, a hypothesis that pupils with negative SJL constitutes a discrete population with typical characteristic features (predominantly male, higher physical activity, and after-school activity scores; less non-school day screen time; and shorter non-school night sleep duration) was raised. Further studies are needed to confirm this hypothesis.