Shedding light on latent pathogenesis and pathophysiology of mental disorders: The potential of iPS cell technology.

Mental disorders are considered as one of the major healthcare issues worldwide owing to their significant impact on the quality of life of patients, causing serious social burdens. However, it is hard to examine the living brain-a source of psychiatric symptoms-at the cellular, subcellular, and molecular levels, which poses difficulty in determining the pathogenesis and pathophysiology of mental disorders. Recently, induced pluripotent stem cell (iPSC) technology has been used as a novel tool for research on mental disorders. We believe that the iPSC-based studies will address the limitations of other research approaches, such as human genome, postmortem brain study, brain imaging, and animal model analysis. Notably, studies using integrated iPSC technology with genetic information have provided significant novel findings to date. This review aimed to discuss the history, current trends, potential, and future of iPSC technology in the field of mental disorders. Although iPSC technology has several limitations, this technology can be used in combination with the other approaches to facilitate studies on mental disorders.

Three-dimensional induction of dorsal, intermediate and ventral spinal cord tissues from human pluripotent stem cells.

The spinal cord contains more than 20 distinct subclasses of neurons that form well-organized neural circuits capable of sensing the environment and generating motor behavior. Although recent studies have described the efficient in vitro generation of spinal motor neurons, the induction of the spinal cord as a whole tissue has not been achieved. In the present study, we demonstrate three-dimensional (3D) induction of dorsal spinal cord-like tissues from human pluripotent stem cells. Our 3D spinal cord induction (3-DiSC) condition recapitulates patterning of the developing dorsal spinal cord and enables the generation of four types of dorsal interneuron marker-positive cell populations. By activating Shh signaling, intermediate and ventral spinal cord-like tissues are successfully induced. After dissociation of these tissues, somatosensory neurons and spinal motor neurons are detected and express neurotransmitters in an in vivo manner. Our approach provides a useful experimental tool for the analysis of human spinal cord development and will contribute to research on the formation and organization of the spinal cord, and its application to regenerative medicine.

Cell-based therapies for Parkinson's disease.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders that is characterized by the loss of A9 midbrain dopaminergic neurons. Recently, cell replacement therapy for PD using iPS cell-derived dopaminergic neurons is attracting public attention, because it is getting closer to clinical application. In this review, we introduce both of the history of cell replacement therapy for PD using fetal ventral mesencephalic tissues and future perspective using iPS cell-derived midbrain dopaminer- gic neurons. Although we must overcome multitude issues including elimination of undifferentiated cells, graft-induced dyskinesia, guarantee of quality of transplanted cells and so on, from there on, we will bring the new generation of stem cell-based cell therapy for PD.

Self-organization of axial polarity, inside-out layer pattern, and species-specific progenitor dynamics in human ES cell-derived neocortex.

Here, using further optimized 3D culture that allows highly selective induction and long-term growth of human ES cell (hESC)-derived cortical neuroepithelium, we demonstrate unique aspects of self-organization in human neocorticogenesis. Self-organized cortical tissue spontaneously forms a polarity along the dorsocaudal-ventrorostral axis and undergoes region-specific rolling morphogenesis that generates a semispherical structure. The neuroepithelium self-forms a multilayered structure including three neuronal zones (subplate, cortical plate, and Cajal-Retzius cell zones) and three progenitor zones (ventricular, subventricular, and intermediate zones) in the same apical-basal order as seen in the human fetal cortex in the early second trimester. In the cortical plate, late-born neurons tend to localize more basally to early-born neurons, consistent with the inside-out pattern seen in vivo. Furthermore, the outer subventricular zone contains basal progenitors that share characteristics with outer radial glia abundantly found in the human, but not mouse, fetal brain. Thus, human neocorticogenesis involves intrinsic programs that enable the emergence of complex neocortical features.

Anti-amyloid ß autoantibodies in cerebral amyloid angiopathy-related inflammation: implications for amyloid-modifying therapies.

OBJECTIVE: Cerebral amyloid angiopathy-related inflammation (CAA-ri) is characterized by vasogenic edema and multiple cortical/subcortical microbleeds, sharing several aspects with the recently defined amyloid-related imaging abnormalities (ARIA) reported in Alzheimer's disease (AD) passive immunization therapies. Herein, we investigated the role of anti-amyloid ß (Aß) autoantibodies in the acute and remission phases of CAA-ri. METHODS: We used a novel ultrasensitive technique on patients from a retrospective multicenter case-control study, and evaluated the anti-Aß autoantibody concentration in the cerebrospinal fluid (CSF) of 10 CAA-ri, 8 CAA, 14 multiple sclerosis, and 25 control subjects. Levels of soluble Aß40, Aß42, tau, P-181 tau, and APOE genotype were also investigated. RESULTS: During the acute phase of CAA-ri, anti-Aß autoantibodies were specifically increased and directly correlated with Aß mobilization, together with augmented tau and P-181 tau. Following clinical and radiological remission, autoantibodies progressively returned to control levels, and both soluble Aß and axonal degeneration markers decreased in parallel. INTERPRETATION: Our data support the hypothesis that the pathogenesis of CAA-ri may be mediated by a selective autoimmune reaction against cerebrovascular Aß, directly related to autoantibody concentration and soluble Aß. The CSF dosage of anti-Aß autoantibodies with the technique here described can thus be proposed as a valid alternative tool for the diagnosis of CAA-ri. Moreover, given the similarities between ARIA developing spontaneously and those observed during immunization trials, anti-Aß autoantibodies can be considered as novel potential biomarkers in future amyloid-modifying therapies for the treatment of AD and CAA.

Significant CMAP decrement by repetitive nerve stimulation is more frequent in median than ulnar nerves of patients with amyotrophic lateral sclerosis.

INTRODUCTION: Several studies have shown a significant amplitude decrement in compound muscle action potentials (CMAPs) on repetitive nerve stimulation (RNS) of muscles involved in amyotrophic lateral sclerosis (ALS). In ALS, muscle wasting preferentially affects the thenar muscles (APB) rather than the hypothenar muscles (ADM). METHODS: We performed RNS studies in the APB and ADM muscles of 32 ALS patients to determine whether the effect of RNS differs between the median and ulnar nerves. RESULTS: The decremental responses to RNS were greater in the APB than in the ADM. Reduced CMAP amplitude was negatively correlated with CMAP decrement in median but not in ulnar nerves. CONCLUSIONS: The greater CMAP decrement in median nerve is attributable to preferential involvement of the APB in the pathophysiology of ALS or some underlying difference in the biology of the two muscles/nerves. Further investigations will better our understanding of the pathophysiology of ALS.

Myasthenic crisis patients who require intensive care unit management.

The purpose of this report was to investigate predictive factors that necessitate intensive care in myasthenic crisis (MC). We retrospectively reviewed MC patients at our institution and compared ICU and ward management groups. Higher MG-ADL scale scores, non-ocular initial symptoms, infection-triggered findings, and higher MGFA classification were observed more frequently in the ICU group. In patients with these prognostic factors, better outcomes may be obtained with early institution of intensive care.

Heatstroke in patients with Parkinson's disease.

We present two Parkinson's disease (PD) patients, who experienced heatstroke. Both patients manifested central nervous system dysfunction with elevated core temperature. Despite adequate lowering of the body temperature, multiorgan-dysfunction syndrome including encephalopathy, rhabdomyolysis, acute renal failure, acute respiratory failure, and disseminated intravascular coagulopathy was noted in one patient, leading to permanent neurologic damage. Because the ensuing multiorgan dysfunction could determine the functional prognosis in heatstroke patients, it is important to provide information about the prevention of heatstroke to patients, who are isolated or are severely disabled in the advanced stages of PD.

Mapping the Ethical Issues of Brain Organoid Research and Application.

In 2008, researchers created human three-dimensional neural tissue - known as the pioneering work of "brain organoids." In recent years, some researchers have transplanted human brain organoids into animal brains for applicational purposes. With these experiments have come many ethical concerns. It is thus an urgent task to clarify what is ethically permissible and impermissible in brain organoid research. This paper seeks (1) to sort out the ethical issues related to brain organoid research and application and (2) to propose future directions for additional ethical consideration and policy debates in the field. Toward (1), this paper first outlines the current state of brain organoid research, and then briefly responds to previously raised related ethical concerns. Looking next at anticipated scientific developments in brain organoid research, we will discuss (i) ethical issues related to in vitro brain organoids, (ii) ethical issues raised when brain organoids form complexes or have relationships with other entities, and (iii) ethical issues of research ethics and governance. Finally, in pursuit of (2), we propose research policies that are mindful of the ethics of brain organoid research and application and also suggest the need for an international framework for research and application of brain organoids.

Cerebral amyloid angiopathy-related inflammation presenting with steroid-responsive higher brain dysfunction: case report and review of the literature.

A 56-year-old man noticed discomfort in his left lower limb, followed by convulsion and numbness in the same area. Magnetic resonance imaging (MRI) showed white matter lesions in the right parietal lobe accompanied by leptomeningeal or leptomeningeal and cortical post-contrast enhancement along the parietal sulci. The patient also exhibited higher brain dysfunction corresponding with the lesions on MRI. Histological pathology disclosed ß-amyloid in the blood vessels and perivascular inflammation, which highlights the diagnosis of cerebral amyloid angiopathy (CAA)-related inflammation. Pulse steroid therapy was so effective that clinical and radiological findings immediately improved.CAA-related inflammation is a rare disease, defined by the deposition of amyloid proteins within the leptomeningeal and cortical arteries associated with vasculitis or perivasculitis. Here we report a patient with CAA-related inflammation who showed higher brain dysfunction that improved with steroid therapy. In cases with atypical radiological lesions like our case, cerebral biopsy with histological confirmation remains necessary for an accurate diagnosis.

Axonal Extensions along Corticospinal Tracts from Transplanted Human Cerebral Organoids.

The reconstruction of lost neural circuits by cell replacement is a possible treatment for neurological deficits after cerebral cortex injury. Cerebral organoids can be a novel source for cell transplantation, but because the cellular composition of the organoids changes along the time course of the development, it remains unclear which developmental stage of the organoids is most suitable for reconstructing the corticospinal tract. Here, we transplanted human embryonic stem cell-derived cerebral organoids at 6 or 10 weeks after differentiation (6w- or 10w-organoids) into mouse cerebral cortices. 6w-organoids extended more axons along the corticospinal tract but caused graft overgrowth with a higher percentage of proliferative cells. Axonal extensions from 10w-organoids were smaller in number but were enhanced when the organoids were grafted 1 week after brain injury. Finally, 10w-organoids extended axons in cynomolgus monkey brains. These results contribute to the development of a cell-replacement therapy for brain injury and stroke.

The Ethics of Cerebral Organoid Research: Being Conscious of Consciousness.

Recently, the use of three-dimensional neural tissues cultured in vitro and called "cerebral organoids" has advanced recapitulation of neural development and disease modeling studies. Along with such advances, cerebral organoid research, and associated concerns call for the elucidation of two points: (1) how cerebral organoid research is currently progressing and the future directions it is likely to take, especially in functional assessment of organoids, and (2) how we should solve ethical issues of possible consciousness in cerebral organoid research. This paper aims first to explore these two issues, and then to present implications and prospects for future cerebral organoid research.

Self-Organized Synchronous Calcium Transients in a Cultured Human Neural Network Derived from Cerebral Organoids.

The cerebrum is a major center for brain function, and its activity is derived from the assembly of activated cells in neural networks. It is currently difficult to study complex human cerebral neuronal network activity. Here, using cerebral organoids, we report self-organized and complex human neural network activities that include synchronized and non-synchronized patterns. Self-organized neuronal network formation was observed following a dissociation culture of human embryonic stem cell-derived cerebral organoids. The spontaneous individual and synchronized activity of the network was measured via calcium imaging, and subsequent analysis enabled the examination of detailed cell activity patterns, providing simultaneous raster plots, cluster analyses, and cell distribution data. Finally, we demonstrated the feasibility of our system to assess drug-inducible dynamic changes of the network activity. The comprehensive functional analysis of human neuronal networks using this system may offer a powerful tool to access human brain function.

Therapeutic effects of combined cell transplantation and locomotor training in rats with brain injury.

Cell-based therapies are attracting attention as alternative therapeutic options for brain damage. In this study, we investigated the therapeutic effect of a combined therapy of cell transplantation and locomotor training by evaluating the neuronal connectivity. We transplanted neural cells derived from the frontal cortex of E14.5 GFP-expressing mice into the frontal lobe of 3-week-old rats with brain injury, followed by treadmill training (TMT) for 14 days. In the TMT(-) group, graft-derived neurites were observed only in the striatum and internal capsule. In contrast, in the TMT(+) group, they were observed in the striatum, internal capsule, and the cerebral peduncle and spinal cord. The length of the longest neurite was significantly longer in the TMT(+) group than in the TMT(-) group. In the TMT(+) group, Synaptophysin+ vesicles on the neuronal fibers around the ipsilateral red nucleus were found, suggesting that neuronal fibers from the grafted cells formed synapses with the host neurons. A functional analysis of motor recovery using the foot fault test showed that, 1 week after the transplantation, the recovery was significantly better in the cell transplantation and TMT group than the cell transplantation only group. The percentage of cells expressing C-FOS was increased in the grafts in the TMT(+) group. In conclusion, TMT promoted neurite extensions from the grafted neural cells, and the combined therapy of cell transplantation and locomotor training might have the potential to promote the functional recovery of rats with brain injury compared to cell transplantation alone.

Generation of Various Telencephalic Regions from Human Embryonic Stem Cells in Three-Dimensional Culture.

In the developing embryo, telencephalon arises from the rostral portion of the neural tube. The telencephalon further subdivides into distinct brain regions along the dorsal-ventral (DV) axis by exogenous patterning signals. Here, we describe a protocol for in vitro generation of various telencephalic regions from human embryonic stem cells (ESCs). Dissociated human ESCs are reaggregated in a low-cell-adhesion 96-well plate and cultured as floating aggregates. Telencephalic neural progenitors are efficiently generated when ESC aggregates are cultured in serum-free medium containing TGFß inhibitor and Wnt inhibitor. In long-term culture, the telencephalic neural progenitors acquire cortical identities and self-organize a stratified cortical structure as seen in human fetal cortex. By treatment with Shh signal, the telencephalic progenitors acquire ventral (subpallial) identities and generate lateral ganglionic eminence (LGE) and medial ganglionic eminence (MGE). In contrast, by treatment with Wnt and BMP signals, their regional identities shift to more dorsal side that generates choroid plexus and medial palllium (hippocampal primordium).

Enhanced Axonal Extension of Subcortical Projection Neurons Isolated from Murine Embryonic Cortex using Neuropilin-1.

The cerebral cortical tissue of murine embryo and pluripotent stem cell (PSC)-derived neurons can survive in the brain and extend axons to the spinal cord. For efficient cell integration to the corticospinal tract (CST) after transplantation, the induction or selection of cortical motor neurons is important. However, precise information about the appropriate cell population remains unclear. To address this issue, we isolated cells expressing Neuropilin-1 (NRP1), a major axon guidance molecule receptor during the early developmental stage, from E14.5 mouse embryonic frontal cortex by fluorescence-activated cell sorting. Aggregates of NRP1+ cells gradually expressed subcortical projection neuron markers, Ctip2 and VGluT1, and axon guidance molecule receptors, Robo1 and deleted in colorectal calcinoma (Dcc), in vitro, suggesting that they contained early-stage subcortical projection neurons. We transplanted NRP1+ cells into the frontal cortex of P2 neonatal mice. Compared with grafts derived from NRP1- or unsorted cells, those derived from NRP1+ cells extended a larger number of axons to the spinal cord along the CST. Our data suggest that sorting NRP1+ cells from the embryonic cerebral cortex enriches subcortical projection neurons to reconstruct the CST.

Generation of functional hippocampal neurons from self-organizing human embryonic stem cell-derived dorsomedial telencephalic tissue.

The developing dorsomedial telencephalon includes the medial pallium, which goes on to form the hippocampus. Generating a reliable source of human hippocampal tissue is an important step for cell-based research into hippocampus-related diseases. Here we show the generation of functional hippocampal granule- and pyramidal-like neurons from self-organizing dorsomedial telencephalic tissue using human embryonic stem cells (hESCs). First, we develop a hESC culture method that utilizes bone morphogenetic protein (BMP) and Wnt signalling to induce choroid plexus, the most dorsomedial portion of the telencephalon. Then, we find that titrating BMP and Wnt exposure allowed the self-organization of medial pallium tissues. Following long-term dissociation culture, these dorsomedial telencephalic tissues give rise to Zbtb20(+)/Prox1(+) granule neurons and Zbtb20(+)/KA1(+) pyramidal neurons, both of which were electrically functional with network formation. Thus, we have developed an in vitro model that recapitulates human hippocampus development, allowing the generation of functional hippocampal granule- and pyramidal-like neurons.

Sporadic juvenile amyotrophic lateral sclerosis caused by mutant FUS/TLS: possible association of mental retardation with this mutation.

We present two cases of patients with juvenile amyotrophic lateral sclerosis (ALS), who had no history of familial ALS. The symptoms of both patients started as weakness of the unilateral upper limb and neck, and extended to bulbar and respiratory weakness in a relatively short period. Of note, the first patient was mentally retarded before the onset of weakness. Fused in sarcoma/translocated in liposarcoma (FUS/TLS) gene analyses revealed mutations of p. G492EfsX527 (c. 1475delG), which is a novel deletion/frameshift mutation, in the first patient and p. R514S mutation (c. 1542G > T) in the second patient. Molecular analysis revealed that the mutant FUS/TLS, especially the deletion/frameshift mutation, showed significant cytoplasmic localization in transfected motor neuron-like cells. Our findings suggest the association of mental retardation with the FUS/TLS mutation. Further investigation, including the effect of FUS/TLS on cognitive function, would aid better understanding of FUS/TLS proteinopathies.

Robust formation and maintenance of continuous stratified cortical neuroepithelium by laminin-containing matrix in mouse ES cell culture.

In the mammalian cortex, the dorsal telencephalon exhibits a characteristic stratified structure. We previously reported that three-dimensional (3D) culture of mouse ES cells (mESCs) can efficiently generate cortical neuroepithelium (NE) and layer-specific cortical neurons. However, the cortical NE generated in this mESC culture was structurally unstable and broke into small neural rosettes by culture day 7, suggesting that some factors for reinforcing the structural integrity were missing. Here we report substantial supporting effects of the extracellular matrix (ECM) protein laminin on the continuous formation of properly polarized cortical NE in floating aggregate culture of mESCs. The addition of purified laminin and entactin (a laminin-associated protein), even at low concentrations, stabilized the formation of continuous cortical NE as well as the maintenance of basement membrane and prevented rosette formation. Treatment with the neutralizing ß1-integrin antibody impaired the continuous NE formation. The stabilized cortical NE exhibited typical interkinetic nuclear migration of cortical progenitors, as seen in the embryonic cortex. The laminin-treated cortical NE maintained a continuous structure even on culture days 12 and 15, and contained ventricular, basal-progenitor, cortical-plate and Cajal-Retzius cell layers. The cortical NE in this culture was flanked by cortical hem-like tissue. Furthermore, when Shh was added, ventral telencephalic structures such as lateral ganglionic eminence-like tissue formed in the region adjacent to the cortical NE. Thus, our results indicate that laminin-entactin ECM promotes the formation of structurally stable telencephalic tissues in 3D ESC culture, and supports the morphogenetic recapitulation of cortical development.

A novel GJB1 frameshift mutation produces a transient CNS symptom of X-linked Charcot-Marie-Tooth disease.

X-linked Charcot-Marie-Tooth disease (CMT1X) is the second most common variant of CMT and is caused by mutations in the GJB1 gene encoding connexin 32. Some CMT1X patients with GJB1 missense mutations have shown transient central nervous system (CNS) symptoms with abnormal brain magnetic resonance imaging (MRI). Herein we report the first case with a novel GJB1 frameshift mutation that associates with a transient CNS symptom. The patient noticed high-arched feet and limited ankle dorsiflexion in early childhood; he transiently developed numbness and paresis of left face and arm, and dysphagia, with abnormal brain MRI. Although the CNS symptoms recovered within several hours without treatment, intravenous immunoglobulin (IVIg) therapy ameliorated progressing symptoms such as those of toe extensor muscles. His mother had been diagnosed with chronic inflammatory demyelinating polyneuropathy (CIDP), and repetitive IVIg treatments had relieved the symptoms. Therefore, inflammation might be involved in the pathophysiology of CMT1X with the GJB1 mutation, while molecular analysis revealed that the mutant GJB1 was more rapidly degraded by the proteasome pathway known as endoplasmic reticulum (ER)-associated degradation.