Publications by authors named "Hideyuki Okano"

833 Publications

Teratoma Formation Assay for Assessing Pluripotency and Tumorigenicity of Pluripotent Stem Cells.

Bio Protoc 2017 Aug 20;7(16):e2518. Epub 2017 Aug 20.

Biomedical Animal Research Laboratory, Institute for Genetic Medicine, Hokkaido University, Hokkaido, Japan.

Pluripotent stem cells such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) form teratomas when transplanted into immunodeficient mice. As teratomas contain all three germ layers (endoderm, mesoderm, ectoderm), teratoma formation assay is widely used as an index of pluripotency (Evans and Kaufman, 1981; Hentze , 2009 ; Gropp , 2012 ). On the other hand, teratoma-forming tumorigenicity also represents a major risk factor impeding potential clinical applications of pluripotent stem cells ( Miura , 2009 ; Okano , 2013 ). Recently, we reported that iPSCs derived from naked mole-rat lack teratoma-forming tumorigenicity when engrafted into the testes of non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice due to an ES cell-expressed Ras (ERAS) and Alternative reading frame (ARF)-dependent tumor-suppression mechanism specific to this species ( Miyawaki , 2016 ). Here, we describe a method for transplanting pluripotent stem cells into the testes of NOD/SCID mice to generate teratomas for assessing the pluripotency and tumorigenicity.
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http://dx.doi.org/10.21769/BioProtoc.2518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8413610PMC
August 2017

First-in-human clinical trial of transplantation of iPSC-derived NS/PCs in subacute complete spinal cord injury: Study protocol.

Regen Ther 2021 Dec 7;18:321-333. Epub 2021 Sep 7.

Department of Physiology, Keio University School of Medicine, Tokyo, Japan.

Introduction: Our group has conducted extensive basic and preclinical studies of the use of human induced pluripotent cell (iPSC)-derived neural stem/progenitor cell (hiPSC-NS/PC) grafts in models of spinal cord injury (SCI). Evidence from animal experiments suggests this approach is safe and effective. We are preparing to initiate a first-in-human clinical study of hiPSC-NS/PC transplantation in subacute SCI.

Setting: NS/PCs were prepared at a Good Manufacturing Practice-grade cell processing facility at Osaka National Hospital using a clinical-grade integration-free hiPSC line established by the iPSC Stock Project organized by the Kyoto University Center for iPS Cell Research and Application. After performing all quality checks, the long-term safety and efficacy of cells were confirmed using immunodeficient mouse models.

Methods: The forthcoming clinical study uses an open-label, single-arm design. The initial follow-up period is 1 year. The primary objective is to assess the safety of hiPSC-NS/PC transplantation in patients with subacute SCI. The secondary objective is to obtain preliminary evidence of its impact on neurological function and quality-of-life outcomes. Four patients with C3/4-Th10 level, complete subacute (within 24 days post-injury) SCI will be recruited. After obtaining consent, cryopreserved cells will be thawed and prepared following a multi-step process including treatment with a γ-secretase inhibitor to promote cell differentiation. A total of 2 × 10 cells will be transplanted into the injured spinal cord parenchyma 14-28 days post-injury. Patients will also receive transient immunosuppression. This study protocol has been reviewed and approved by the Certified Committee for Regenerative Medicine and the Japanese Ministry of Health, Labor and Welfare (University Hospital Medical Information Network Clinical Trials Registry [UMIN-CTR] number, UMIN000035074; Japan Registry of Clinical Trials [jRCT] number, jRCTa031190228).

Discussion/conclusion: We plan to start recruiting a patient as soon as the COVID-19 epidemic subsides. The primary focus of this clinical study is safety, and the number of transplanted cells may be too low to confirm efficacy. After confirming safety, a dose-escalation study is planned.
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http://dx.doi.org/10.1016/j.reth.2021.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8427225PMC
December 2021

Markerless analysis of hindlimb kinematics in spinal cord-injured mice through deep learning.

Neurosci Res 2021 Sep 8. Epub 2021 Sep 8.

Department of Physiology, Keio University School of Medicine, Tokyo, Japan. Electronic address:

Rodent models are commonly used to understand the underlying mechanisms of spinal cord injury (SCI). Kinematic analysis, an important technique to measure dysfunction of locomotion after SCI, is generally based on the capture of physical markers placed on bony landmarks. However, marker-based studies face significant experimental hurdles such as labor-intensive manual joint tracking, alteration of natural gait by markers, and skin error from soft tissue movement on the knee joint. Although the pose estimation strategy using deep neural networks can solve some of these issues, it remains unclear whether this method is adaptive to SCI mice with abnormal gait. In the present study, we developed a deep learning based markerless method of 2D kinematic analysis to automatically track joint positions. We found that a relatively small number (< 200) of manually labeled video frames was sufficient to train the network to extract trajectories. The mean test error was on average 3.43 pixels in intact mice and 3.95 pixels in SCI mice, which is comparable to the manual tracking error (3.15 pixels, less than 1 mm). Thereafter, we extracted 30 gait kinematic parameters and found that certain parameters such as step height and maximal hip joint amplitude distinguished intact and SCI locomotion.
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http://dx.doi.org/10.1016/j.neures.2021.09.001DOI Listing
September 2021

Modeling human congenital disorders with neural crest developmental defects using patient-derived induced pluripotent stem cells.

Regen Ther 2021 Dec 24;18:275-280. Epub 2021 Aug 24.

Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan.

The neural crest is said to be the fourth germ layer in addition to the ectoderm, mesoderm and endoderm because of its ability to differentiate into a variety of cells that contribute to the various tissues of the vertebrate body. Neural crest cells (NCCs) can be divided into three functional groups: cranial NCCs, cardiac NCCs and trunk NCCs. Defects related to NCCs can contribute to a broad spectrum of syndromes known as neurocristopathies. Studies on the neural crest have been carried out using animal models such as , chicks, and mice. However, the precise control of human NCC development has not been elucidated in detail due to species differences. Using induced pluripotent stem cell (iPSC) technology, we developed an disease model of neurocristopathy by inducing the differentiation of patient-derived iPSCs into NCCs and/or neural crest derivatives. It is now possible to address complicated questions regarding the pathogenetic mechanisms of neurocristopathies by characterizing cellular biological features and transcriptomes and by transplanting patient-derived NCCs . Here, we provide some examples that elucidate the pathophysiology of neurocristopathies using disease modeling via iPSCs.
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http://dx.doi.org/10.1016/j.reth.2021.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8390449PMC
December 2021

Developmental dysregulation of excitatory-to-inhibitory GABA-polarity switch may underlie schizophrenia pathology: A monozygotic-twin discordant case analysis in human iPS cell-derived neurons.

Neurochem Int 2021 Sep 6;150:105179. Epub 2021 Sep 6.

Department of Psychiatry, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara, 634-8522, Japan.

Schizophrenia is a major psychiatric disorder, but the molecular mechanisms leading to its initiation or progression remain unclear. To elucidate the pathophysiology of schizophrenia, we used an in vitro neuronal cell culture model involving human induced pluripotent stem cells (hiPSCs) derived from a monozygotic-twin discordant schizophrenia pair. The cultured neurons differentiated from hiPSCs were composed of a mixture of glutamatergic excitatory neurons and gamma aminobutyric acid (GABA)ergic inhibitory neurons. In the electrophysiological analysis, a different pattern of spontaneous neuronal activity was observed under the condition without any stimulants. The frequency of spontaneous excitatory post-synaptic currents (sEPSCs) was significantly higher in the hiPSC-derived neurons of the patient with schizophrenia than in the control sibling at day-in-vitro 30. However, the synaptic formation was not different between the patient with schizophrenia and the control sibling during the same culture period. To explain underlying mechanisms of higher excitability of presynaptic cells, we focused on the potassium-chloride co-transporter KCC2, which contributes to excitatory-to-inhibitory GABA polarity switch in developing neurons. We also revealed the altered expression pattern of KCC2 in hiPSC-derived neurons from the patient with schizophrenia, which could contribute to understanding the pathology of schizophrenia in the developing nervous system.
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http://dx.doi.org/10.1016/j.neuint.2021.105179DOI Listing
September 2021

Comparison of Drug Availability in the Inner Ear After Oral, Transtympanic, and Combined Administration.

Front Neurol 2021 19;12:641593. Epub 2021 Aug 19.

Department of Otolaryngology Head and Neck Surgery, Second Affiliated Hospital, Xi'an Jiaotong University, Xi'an, China.

Although combination of oral and transtympanic drug therapy (CT) has been proved more effective and safer for idiopathic sudden sensorineural hearing loss (ISSNHL) by some clinical trials, there are few laboratory researches on the pharmacokinetics in the inner ear following CT on account of structural limitations of the inner ear. The aim of the present study was to investigate the pharmacokinetic behaviors of CT in the inner ear of mice. Eighteen transgenic mice which express luciferase in cochlear spiral ganglion cells were divided into oral administration (OR) group, transtympanic injection route (TT) group and CT group, and luciferin was delivered into the inner ear of these mice through oral, transtympanic or combined routes, respectively. A new imaging system was used to observe luciferin/luciferase signals and the compare the pharmacokinetics of different administration routes in the inner ear of mice. Bioluminescence signals were observed in the inner ear 3.3 ± 2.6 min after CT, significantly earlier than that of OR group (15.8 ± 7.4 min). CT owned the longest reaching-peak time and largest area under the curve (AUC) among three groups. Compared to TT, CT had longer biological half-life and higher AUC value, but did not displayed stronger peak value. There were significant differences in the peak values between OR group and TT group and between OR group and CT group. This study suggests that the OR route is less effective than the TT or CT route, and combination of OR and TT can deliver more drugs into the inner ear and confer a longer therapeutic window, but cannot increase drug intensity.
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http://dx.doi.org/10.3389/fneur.2021.641593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8420880PMC
August 2021

NEAT1 lncRNA and amyotrophic lateral sclerosis.

Neurochem Int 2021 Sep 2;150:105175. Epub 2021 Sep 2.

Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo, Japan. Electronic address:

Amyotrophic lateral sclerosis (ALS) is a representative neurological disease that is known to devastate entire motor neurons within a period of just a few years. Discoveries of the specific pathologies of relevant RNA-binding proteins, including TAR DNA-binding protein-43 (TDP-43) and fused in sarcoma/translocated in liposarcoma (FUS/TLS), and the causative genes of both familial and sporadic ALS have provided crucial information that could lead to a cure. In recent ALS research the GGGGCC-repeat expansion in the C9orf72 gene was identified as one of the most important pathological findings, suggesting the significance of both nuclear dysfunction due to dipeptide repeat proteins (DPRs) and RNA toxicity (such as pathological alterations of non-coding RNAs). In research on model animals carrying ALS-related molecules, the determination of whether a factor is protective or toxic has been controversial. Herein, we review the findings regarding NEAT1 RNA and C9orf72 GGGGCC repeats associated with ALS, from the viewpoint of conversion from the protective stage in the nucleus in early-phase ALS to late-phase induction of cell death. This review will provide insights for the development of RNA effectors as novel ALS treatments.
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http://dx.doi.org/10.1016/j.neuint.2021.105175DOI Listing
September 2021

Transplantation of iPSC-derived corneal endothelial substitutes in a monkey corneal edema model.

Stem Cell Res 2021 Aug 9;55:102497. Epub 2021 Aug 9.

Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan. Electronic address:

Objective: In order to provide regenerative therapy for millions of patients suffering from corneal blindness globally, we derived corneal endothelial cell substitute (CECSi) cells from induced pluripotent stem cells (iPSCs) to treat corneal edema due to endothelial dysfunction (bullous keratopathy).

Methods And Results: We developed an efficient xeno-free protocol to produce CECSi cells from both research grade (Ff-MH09s01 and Ff-I01s04) and clinical grade (QHJI01s04) iPSCs. CECSi cells formed a hexagonal confluent monolayer with Na, K-ATPase alpha 1 subunit expression (ATP1A1), tight junctions, N-cadherin adherence junction formation, and nuclear PITX2 expression, which are all characteristics of corneal endothelial cells. CECSi cells can be cryopreserved, and thawed CECSi cell suspensions also expressed N-cadherin and ATP1A1. Residual undifferentiated iPSCs in QHJI01s04-derived CECSi cells was below 0.01%. Frozen stocks of Ff-I01s04- and QHJI01s04-derived CECSi cells were transported, thawed and transplanted into a monkey corneal edema model. CECSi-transplanted eyes significantly reduced corneal edema compared to control group.

Conclusion: Our results show a promising approach to provide bullous keratopathy patients with an iPS-cell-based cell therapy to recover useful vision.
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http://dx.doi.org/10.1016/j.scr.2021.102497DOI Listing
August 2021

Human iPS Cell-Derived Cell Aggregates Exhibited Dermal Papilla Cell Properties in Three-Dimensional Assemblage Mimicking Hair Follicle Structures.

Front Cell Dev Biol 2021 2;9:590333. Epub 2021 Aug 2.

Department of Dermatology, Kyorin University Faculty of Medicine, Tokyo, Japan.

Current approaches for human hair follicle (HF) regeneration mostly adopt cell-autonomous tissue reassembly in a permissive murine intracorporeal environment. This, together with the limitation in human-derived trichogenic starting materials, potentially hinders the bioengineering of human HF structures, especially for the drug discovery and treatment of hair loss disorders. In this study, we attempted to reproduce the anatomical relationship between an epithelial main body and the dermal papilla (DP) within HF by three-dimensionally assembling columnarly molded human keratinocytes (KCs) and the aggregates of DP cells and evaluated how HF characteristics were reproduced in the constructs. The replaceability of human-induced pluripotent stem cell (hiPSC)-derived DP substitutes was assessed using the aforementioned reconstruction assay. Human DP cell aggregates were embedded into Matrigel as a cluster. Subsequently, highly condensed human KCs were cylindrically injected onto DP spheroids. After 2-week culture, the structures visually mimicking HFs were obtained. KC-DP constructs partially reproduced HF microanatomy and demonstrated differential keratin (KRT) expression pattern in HFs: KRT14 in the outermost part and KRT13, KRT17, and KRT40, respectively, in the inner portion of the main body. KC-DP constructs tended to upregulate HF-related genes, , , , , and . Next, DP substitutes were prepared by exposing hiPSC-derived mesenchymal cells to retinoic acid and subsequently to WNT, BMP, and FGF signal activators, followed by cell aggregation. The resultant hiPSC-derived DP substitutes (iDPs) were combined with KCs in the invented assay. KC-iDP constructs morphologically resemble KC-DP constructs and analogously mimicked KRT expression pattern in HF. iDP in the constructs expressed DP-related markers, such as vimentin and versican. Intriguingly, KC-iDP constructs more intensely expressed , , and , which were stepwisely upregulated by the addition of WNT ligand and the mixture of WNT, SHH, and EDA signaling activators, supporting the idea that iDP exhibited biological properties analogous to DP cell aggregates in the constructs . These preliminary findings suggested the possibility of regenerating DP equivalents with hair-inductive capacity using hiPSC-derived cell composites, which potentially reduce the necessity of human tissue-derived trichogenic cell subset and eventually allow xeno-free bioengineering of human HFs.
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http://dx.doi.org/10.3389/fcell.2021.590333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8365839PMC
August 2021

Treadmill training based on the overload principle promotes locomotor recovery in a mouse model of chronic spinal cord injury.

Exp Neurol 2021 Nov 8;345:113834. Epub 2021 Aug 8.

Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Electronic address:

Rehabilitative treatment, including treadmill training, is considered an important strategy for restoring motor function after spinal cord injury (SCI). However, many unexplained problems persist regarding the appropriate rehabilitative method and the mechanism underlying the beneficial effects of rehabilitation. Moreover, only a few preclinical studies have been performed on rehabilitative interventions for chronic SCI, although most patients have chronic injuries. In fact, several preclinical studies reported that rehabilitative training was less effective when applied during the chronic phase than when applied sooner. While numerous studies have examined the effects of treadmill training during the subacute phase, the training conditions vary considerably among preclinical reports. Therefore, establishing a standard training protocol is essential for achieving beneficial rehabilitation effects at the chronic stage. Since the difficulty of applying an appropriate training load hinders training at constant speeds, it is important to adjust the training intensity in accordance with the exercise tolerance of an individual animal to provide further functional recovery benefits. Here, we created a novel quadrupedal treadmill training protocol based on the overload principle for mice with incomplete thoracic SCI. We subjected SCI model mice to rehabilitative training according to the protocol for two consecutive weeks starting at 42 days after injury. We examined the treadmill speeds at which the mice were able to run based on the severity of paresis and investigated the impact of the protocol on functional recovery. Assessment of running speed changes during the treadmill training period revealed faster treadmill speeds for mice with mild paresis than for those with severe paresis. The training parameters, including the speed and distance traveled, were positively correlated with the changes in motor function. These results suggest that the most suitable running speed during treadmill training differs according to the level of motor dysfunction and that running longer distances has a positive impact on motor functional recovery. Based on this established protocol, we compared functional and histological results between the chronic SCI groups with and without rehabilitation. The gait analyses showed significantly better functional improvement in the rehabilitation group than in the nonrehabilitation group. Histological analyses revealed that the BDNF- and VGLUT1-positive areas of lumbar enlargement were significantly increased in the rehabilitation group. These findings implied that rehabilitation promoted not only motor performance but also motor control, including forelimb-hindlimb coordination, even in chronic SCI, resulting in functional improvement by treadmill training alone. Therefore, rehabilitative training based on the overload principle appears to be one of the appropriate treatment options for incomplete thoracic SCI, and evidence of its efficacy exists in actual clinical settings.
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http://dx.doi.org/10.1016/j.expneurol.2021.113834DOI Listing
November 2021

Dynamic Spatiotemporal Expression Changes in Connexins of the Developing Primate's Cochlea.

Genes (Basel) 2021 Jul 16;12(7). Epub 2021 Jul 16.

Department of Otorhinolaryngology, Head and Neck Surgery, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo 160-8582, Japan.

Connexins are gap junction components that are essential for acquiring normal hearing ability. Up to 50% of congenital, autosomal-recessive, non-syndromic deafness can be attributed to variants in , the gene that encodes connexin 26. Gene therapies modifying the expression of connexins are a feasible treatment option for some patients with genetic hearing losses. However, the expression patterns of these proteins in the human fetus are not fully understood due to ethical concerns. Recently, the common marmoset was used as a primate animal model for the human fetus. In this study, we examined the expression patterns of connexin 26 and connexin 30 in the developing cochlea of this primate. Primate-specific spatiotemporal expression changes were revealed, which suggest the existence of primate-specific control of connexin expression patterns and specific functions of these gap junction proteins. Moreover, our results indicate that treatments for connexin-related hearing loss established in rodent models may not be appropriate for human patients, underscoring the importance of testing these treatments in primate models before applying them in human clinical trials.
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http://dx.doi.org/10.3390/genes12071082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8307058PMC
July 2021

A 50 Hz magnetic field affects hemodynamics, ECG and vascular endothelial function in healthy adults: A pilot randomized controlled trial.

PLoS One 2021 5;16(8):e0255242. Epub 2021 Aug 5.

Advanced Institute of Innovative Technology, Saitama University, Saitama, Japan.

Application of exposure to 50/60 Hz magnetic fields (MFs) has been conducted in the treatment of muscle pain and fatigue mainly in Japan. However, whether MFs could increase blood flow leading to muscle fatigue recovery has not been sufficiently tested. We investigated the acute effects of a 50 Hz sinusoidal MF at Bmax 180 mT on hemodynamics, electrocardiogram, and vascular endothelial function in healthy young men. Three types of regional exposures to a 50 Hz MF, i.e., forearm, upper arm, or neck exposure to MF were performed. Participants who received three types of real MF exposures had significantly increased ulnar arterial blood flow velocity compared to the sham exposures. Furthermore, after muscle loading exercise, MF exposure recovered hemoglobin oxygenation index values faster and higher than sham exposure from the loading condition. Moreover, participants who received real MF exposure in the neck region had significantly increased parasympathetic high-frequency activity relative to the sham exposure. The MF exposure in the upper arm region significantly increased the brachial artery flow-mediated dilation compared to the sham exposure. Computer simulations of induced in situ electric fields indicated that the order-of-magnitude estimates of the peak values were 100-500 mV/m, depending on the exposure conditions. This study provides the first evidence that a 50 Hz MF can activate parasympathetic activity and thereby lead to increase vasodilation and blood flow via a nitric oxide-dependent mechanism. Trial registration: UMIN Clinical Trial Registry (CTR) UMIN000038834. The authors confirm that all ongoing and related trials for this drug/intervention are registered.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0255242PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8341886PMC
August 2021

Optical manipulation of local cerebral blood flow in the deep brain of freely moving mice.

Cell Rep 2021 Jul;36(4):109427

Department of Neuropsychiatry, Keio University School of Medicine, Tokyo 160-8582, Japan. Electronic address:

An artificial tool for manipulating local cerebral blood flow (CBF) is necessary for understanding how CBF controls brain function. Here, we generate vascular optogenetic tools whereby smooth muscle cells and endothelial cells express optical actuators in the brain. The illumination of channelrhodopsin-2 (ChR2)-expressing mice induces a local reduction in CBF. Photoactivated adenylyl cyclase (PAC) is an optical protein that increases intracellular cyclic adenosine monophosphate (cAMP), and the illumination of PAC-expressing mice induces a local increase in CBF. We target the ventral striatum, determine the temporal kinetics of CBF change, and optimize the illumination intensity to confine the effects to the ventral striatum. We demonstrate the utility of this vascular optogenetic manipulation in freely and adaptively behaving mice and validate the task- and actuator-dependent behavioral readouts. The development of vascular optogenetic animal models will help accelerate research linking vasculature, circuits, and behavior to health and disease.
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http://dx.doi.org/10.1016/j.celrep.2021.109427DOI Listing
July 2021

Current Status of and Perspectives on the Application of Marmosets in Neurobiology.

Authors:
Hideyuki Okano

Annu Rev Neurosci 2021 07 2;44:27-48. Epub 2020 Dec 2.

Department of Physiology, Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan; email:

The common marmoset (), a small New World primate, is receiving substantial attention in the neuroscience and biomedical science fields because its anatomical features, functional and behavioral characteristics, and reproductive features and its amenability to available genetic modification technologies make it an attractive experimental subject. In this review, I outline the progress of marmoset neuroscience research and summarize both the current status (opportunities and limitations) of and the future perspectives on the application of marmosets in neuroscience and disease modeling.
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http://dx.doi.org/10.1146/annurev-neuro-030520-101844DOI Listing
July 2021

Involvement of ferroptosis in human motor neuron cell death.

Biochem Biophys Res Commun 2021 Aug 7;566:24-29. Epub 2021 Jun 7.

Department of Physiology, Keio University School of Medicine, Tokyo, 160-8582, Japan; Division of Neurobiology and Anatomy, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, 951-8510, Japan. Electronic address:

Ferroptosis was recently defined as a novel type of programmed cell death depending on iron and lipid peroxidation. It is biologically different from other types of cell death such as apoptosis. While the involvement of ferroptosis in cancer, patient and animal model have been intensely studied, ferroptosis in human motor neuron model is still clearly unknown. Here we carefully assessed ferroptosis using human iPS cell-derived motor neuron (hiMNs). We found that almost all hiMNs died by the treatment of glutathione peroxidase 4 (GPX4) inhibitors. Importantly, the cell death was rescued by one antioxidant, vitamin E acetate, iron chelators and lipid peroxidase inhibitors with high dynamic ranges. Finally, these data clearly indicated that ferroptosis constitutively occurs in hiMNs, suggesting the possibility that it might play a biologically and pathologically important roles in motor neuron death such as motor neuron disease (MND)/Amyotrophic lateral sclerosis (ALS).
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http://dx.doi.org/10.1016/j.bbrc.2021.05.095DOI Listing
August 2021

Establishment of an induced pluripotent stem cell line from a female domestic ferret (Mustela putorius furo) with an X chromosome instability.

Stem Cell Res 2021 05 11;53:102385. Epub 2021 May 11.

Department of Physiology, Keio University School of Medicine, Tokyo, Japan; Laboratory for Marmoset Neural Architecture, RIKEN Center for Brain Science, Saitama, Japan. Electronic address:

The domestic ferret (ferret; Mustela putorius furo) is an important animal model for neuroscience and preclinical/veterinary medicine owing to its highly developed cerebral cortex and susceptibility to avian influenza and corona viruses. Nevertheless, there is a lack of in vitro ferret models, since immortal cell lines including induced pluripotent stem cells (iPSCs) of ferrets have been scarce. In this study, we established an iPSC line from ferret skin fibroblasts. The established iPSC line, fiPS-1, showed standard characteristics of pluripotency, but its X chromosome was unstable. Collectively, the present study provides a useful resource for in vitro model using the ferret.
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http://dx.doi.org/10.1016/j.scr.2021.102385DOI Listing
May 2021

Establishing an induced pluripotent stem cell line from neonatal common marmoset fibroblasts by an all-in-one episomal vector approach.

Stem Cell Res 2021 05 30;53:102380. Epub 2021 Apr 30.

Department of Physiology, School of Medicine, Keio University, Tokyo, Japan; Laboratory for Marmoset Neural Architecture, RIKEN Center for Brain Science, Saitama, Japan. Electronic address:

Epstein-Barr virus (EBV)-based episomal vector system enables persistent transgene expression, which is advantageous for efficient derivation of transgene-free induced pluripotent stem cells (iPSCs) without viral transduction. Here, we report establishment of an iPSC line from somatic fibroblasts of a neonatal common marmoset monkey (marmoset; Callithrix jacchus) using an all-in-one episomal vector that we newly developed. The established iPSC line, named NM-iPS, showed standard characteristics of pluripotency such as pluripotency-related marker expression, three germ layer differentiation, and normal karyotype (2n = 46). The novel iPSC line would be a useful resource for stem cell research using non-human primates.
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http://dx.doi.org/10.1016/j.scr.2021.102380DOI Listing
May 2021

Non-viral derivation of a transgene-free induced pluripotent stem cell line from a male beagle dog.

Stem Cell Res 2021 05 29;53:102375. Epub 2021 Apr 29.

Department of Physiology, School of Medicine, Keio University, Tokyo, Japan. Electronic address:

We previously reported the non-viral derivation of transgene-free induced pluripotent stem cells (iPSCs) from somatic fibroblasts of a female beagle dog using an optimized induction medium and integration-free episomal vectors. Here, we report novel derivation of a male canine iPSC line OF35Y-iPS, which showed standard characteristics of pluripotency such as a strong gene expression profile of pluripotency markers, differentiation potential into all three germ layers, and normal karyotype (78XY). Furthermore, we demonstrated targeted integration of 2A-EGFP into the canine NANOS3 locus. The novel iPSC line would be a useful resource for stem cell research and regenerative veterinary medicine.
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http://dx.doi.org/10.1016/j.scr.2021.102375DOI Listing
May 2021

Reduced PHOX2B stability causes axonal growth impairment in motor neurons with TARDBP mutations.

Stem Cell Reports 2021 Jun 27;16(6):1527-1541. Epub 2021 May 27.

Department of Neurology, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.stemcr.2021.04.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8190591PMC
June 2021

MeCP2 controls neural stem cell fate specification through miR-199a-mediated inhibition of BMP-Smad signaling.

Cell Rep 2021 May;35(7):109124

Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan. Electronic address:

Rett syndrome (RTT) is a severe neurological disorder, with impaired brain development caused by mutations in MECP2; however, the underlying mechanism remains elusive. We know from previous work that MeCP2 facilitates the processing of a specific microRNA, miR-199a, by associating with the Drosha complex to regulate neuronal functions. Here, we show that the MeCP2/miR-199a axis regulates neural stem/precursor cell (NS/PC) differentiation. A shift occurs from neuronal to astrocytic differentiation of MeCP2- and miR-199a-deficient NS/PCs due to the upregulation of a miR-199a target, Smad1, a downstream transcription factor of bone morphogenetic protein (BMP) signaling. Moreover, miR-199a expression and treatment with BMP inhibitors rectify the differentiation of RTT patient-derived NS/PCs and development of brain organoids, respectively, suggesting that facilitation of BMP signaling accounts for the impaired RTT brain development. Our study illuminates the molecular pathology of RTT and reveals the MeCP2/miR-199a/Smad1 axis as a potential therapeutic target for RTT.
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http://dx.doi.org/10.1016/j.celrep.2021.109124DOI Listing
May 2021

Neurological pathogenesis of SARS-CoV-2 (COVID-19): from virological features to clinical symptoms.

Inflamm Regen 2021 May 7;41(1):15. Epub 2021 May 7.

Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan.

Since the worldwide outbreak of coronavirus disease 2019 (COVID-19) in 2020, various research reports and case reports have been published. It has been found that COVID-19 causes not only respiratory disorders but also thrombosis and gastrointestinal disorders, central nervous system (CNS) disorders, and peripheral neuropathy. Compared to other disorders, there are low number of research reports and low number of summaries on COVID-19-related neural disorders. Therefore, focusing on neural disorders, we outline both basic research and clinical manifestations of COVID-19-related neural disorders.
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http://dx.doi.org/10.1186/s41232-021-00165-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8103065PMC
May 2021

Impaired neuronal activity and differential gene expression in STXBP1 encephalopathy patient iPSC-derived GABAergic neurons.

Hum Mol Genet 2021 Jun;30(14):1337-1348

Department of Pediatrics, School of Medicine, Fukuoka University, Fukuoka 814-0180, Japan.

Syntaxin-binding protein 1 (STXBP1; also called MUNC18-1), encoded by STXBP1, is an essential component of the molecular machinery that controls synaptic vesicle docking and fusion. De novo pathogenic variants of STXBP1 cause a complex set of neurological disturbances, namely STXBP1 encephalopathy (STXBP1-E) that includes epilepsy, neurodevelopmental disorders and neurodegeneration. Several animal studies have suggested the contribution of GABAergic dysfunction in STXBP1-E pathogenesis. However, the pathophysiological changes in GABAergic neurons of these patients are still poorly understood. Here, we exclusively generated GABAergic neurons from STXBP1-E patient-derived induced pluripotent stem cells (iPSCs) by transient expression of the transcription factors ASCL1 and DLX2. We also generated CRISPR/Cas9-edited isogenic iPSC-derived GABAergic (iPSC GABA) neurons as controls. We demonstrated that the reduction in STXBP1 protein levels in patient-derived iPSC GABA neurons was slight (approximately 20%) compared to the control neurons, despite a 50% reduction in STXBP1 mRNA levels. Using a microelectrode array-based assay, we found that patient-derived iPSC GABA neurons exhibited dysfunctional maturation with reduced numbers of spontaneous spikes and bursts. These findings reinforce the idea that GABAergic dysfunction is a crucial contributor to STXBP1-E pathogenesis. Moreover, gene expression analysis revealed specific dysregulation of genes previously implicated in epilepsy, neurodevelopment and neurodegeneration in patient-derived iPSC GABA neurons, namely KCNH1, KCNH5, CNN3, RASGRF1, SEMA3A, SIAH3 and INPP5F. Thus, our study provides new insights for understanding the biological processes underlying the widespread neuropathological features of STXBP1-E.
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http://dx.doi.org/10.1093/hmg/ddab113DOI Listing
June 2021

Amyloid β (Aβ) ELISA of Human iPSC-Derived Neuronal Cultures.

Methods Mol Biol 2021 May 7. Epub 2021 May 7.

Department of Physiology, Keio University, School of Medicine, Tokyo, Japan.

Amyloid β (Aβ) peptides are the main component of the characteristic insoluble deposits in brain parenchyma and small blood vessels in the patients afflicted with Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). These small peptides are attributed to the pathogenesis of both AD and CAA, suggesting an important index for disease stage and progression. In the brain tissue, Aβs are released mainly from neuronal cells into extracellular space. Here, we describe a step-by-step protocol to measure Aβs secreted from human pluripotent stem cell-derived neuronal cells.
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http://dx.doi.org/10.1007/7651_2021_407DOI Listing
May 2021

DCTN1 Binds to TDP-43 and Regulates TDP-43 Aggregation.

Int J Mol Sci 2021 Apr 13;22(8). Epub 2021 Apr 13.

Department of Neurology, Faculty of Medicine, Fukuoka University, Fukuoka 814-0180, Japan.

A common pathological hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis, is cytoplasmic mislocalization and aggregation of nuclear RNA-binding protein TDP-43. Perry disease, which displays inherited atypical parkinsonism, is a type of TDP-43 proteinopathy. The causative gene encodes the largest subunit of the dynactin complex. Dynactin associates with the microtubule-based motor cytoplasmic dynein and is required for dynein-mediated long-distance retrograde transport. Perry disease-linked missense mutations (e.g., p.G71A) reside within the CAP-Gly domain and impair the microtubule-binding abilities of DCTN1. However, molecular mechanisms by which such mutations cause TDP-43 proteinopathy remain unclear. We found that DCTN1 bound to TDP-43. Biochemical analysis using a panel of truncated mutants revealed that the DCTN1 CAP-Gly-basic supradomain, dynactin domain, and C-terminal region interacted with TDP-43, preferentially through its C-terminal region. Remarkably, the p.G71A mutation affected the TDP-43-interacting ability of DCTN1. Overexpression of DCTN1, the dynactin-domain fragment, or C-terminal fragment, but not the CAP-Gly-basic fragment, induced cytoplasmic mislocalization and aggregation of TDP-43, suggesting functional modularity among TDP-43-interacting domains of DCTN1. We thus identified DCTN1 as a new player in TDP-43 cytoplasmic-nuclear transport, and showed that dysregulation of DCTN1-TDP-43 interactions triggers mislocalization and aggregation of TDP-43, thus providing insights into the pathological mechanisms of Perry disease and other TDP-43 proteinopathies.
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http://dx.doi.org/10.3390/ijms22083985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8070438PMC
April 2021

A new approach to analysis of intracellular proteins and subcellular localization using cellprofiler and imageJ in combination.

Methods 2021 Apr 26. Epub 2021 Apr 26.

Department of Physiology, Keio University School of Medicine, Tokyo, Japan. Electronic address:

Analytical pipeline, which is used for various analysis application, of CellProfiler, an open-source software for cell imaging analysis, is very important. In the present study, to examine whether intracellular proteins can be discriminated using a combination of CellProfiler and ImageJ, we analyzed neuroblastoma and monocytic cell lines, and disease-specific induced pluripotent stem cell (iPSC)-derived neurons. This revealed that scattered puncta of Rab7 and transferrin in neuroblastoma lines were clearly detectable by created analytical pipelines in CellProfiler. We then constructed pipelines for measuring the distance from the center of the nucleus to allow investigation of the intracellular localization of Rab7 or transferrin. Using CellProfiler and ImageJ in combination, we confirmed that our pipelines were applicable both quantitatively and objectively to analysis of membrane trafficking of proteins such as Rab proteins and transferrin. In addition, when applied to quantitative measurement of phagocytosis, our pipelines clearly detected monocytic cell lines that had engulfed bioparticles. Finally, we developed new pipelines for analysis of disease phenotype using iPSCs from a patient with familial Parkinson's disease (PD), harboring the I2020T LRRK2 mutation (PARK8). These were able to successfully detect Rab5 puncta and Rab7 puncta in PARK8 patient iPSC-derived neurons. Interestingly, in long-term culture, we found that the numbers of Rab7 puncta in a single PARK8 patient iPSC-derived neurons were lower than that of control iPSC-derived neurons. On the other hands, at 14 days in vitro, the numbers of Rab5 puncta in PARK8 patient iPSC-derived neurons were lower than those of isogenic iPSC-derived neurons, but not Rab7 puncta. Furthermore, Rab5 puncta of PARK8 patient iPSC-derived neurons exhibited distinct localization pattern relative to isogenic iPSC-derived neurons. These present results suggest that this new analytical tool can be used as a supporting method for quantification of intracellular protein.
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http://dx.doi.org/10.1016/j.ymeth.2021.04.019DOI Listing
April 2021

Association among extracellular superoxide dismutase genotype, plasma concentration, and comorbidity in the very old and centenarians.

Sci Rep 2021 Apr 20;11(1):8539. Epub 2021 Apr 20.

Center for Supercentenarian Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan.

Superoxide dismutase 3 (SOD3), an antioxidant enzyme, is known as extracellular SOD (EC-SOD) because it is the predominant form in extracellular fluids. The diversity of plasma EC-SOD concentration is associated with the SOD3 p.R231G missense variant genotype. To clarify the association among SOD3 genotype, plasma EC-SOD concentration, and comorbidity in Oldest Old, we analyzed genome-wide associations with plasma EC-SOD concentration and associations between EC-SOD concentration and medical history classified by the SOD3 genotype in the Very Old (85-99 years old, n = 505) and Centenarians (over 100 years old, n = 595). The results revealed that SOD3 p.R231G was the most significant variant associated with plasma EC-SOD concentration. Although no significant difference was observed in medical histories between the SOD3 p.R231G variant non-carriers and carriers, higher EC-SOD concentration in plasma of SOD3 p.R231G variant non-carriers was associated with a high odds ratio for chronic kidney disease (OR = 2.70, 95% CI = 1.98-3.72) and low odds ratio for diabetes mellitus (DM) (OR = 0.61, 95% CI = 0.39-0.95). Comparison with 11 plasma biomarkers for age-related disease showed that plasma EC-SOD concentration correlated with adiponectin and estimated glomerular filtration rate with creatinine correction; therefore, we deduced that EC-SOD co-operates with adiponectin and possesses beneficial functions for DM in the Oldest Old.
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http://dx.doi.org/10.1038/s41598-021-87982-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058336PMC
April 2021

Identification of hub molecules of FUS-ALS by Bayesian gene regulatory network analysis of iPSC model: iBRN.

Neurobiol Dis 2021 07 20;155:105364. Epub 2021 Apr 20.

Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan. Electronic address:

Fused in sarcoma/translated in liposarcoma (FUS) is a causative gene of amyotrophic lateral sclerosis (ALS). Mutated FUS causes accumulation of DNA damage and cytosolic stress granule (SG) formation, thereby motor neuron (MN) death. However, key molecular aetiology remains unclear. Here, we applied a novel platform technology, iBRN, "Non- biased" Bayesian gene regulatory network analysis based on induced pluripotent stem cell (iPSC)-derived cell model, to elucidate the molecular aetiology using transcriptome of iPSC-derived MNs harboring FUS. iBRN revealed "hub molecules", which strongly influenced transcriptome network, such as miR-125b-5p-TIMELESS axis and PRKDC for the molecular aetiology. Next, we confirmed miR-125b-5p-TIMELESS axis in FUS MNs such that miR-125b-5p regulated several DNA repair-related genes including TIMELESS. In addition, we validated both introduction of miR-125b-5p and knocking down of TIMELESS caused DNA damage in the cell culture model. Furthermore, PRKDC was strongly associated with FUS mis-localization into SGs by DNA damage under impaired DNA-PK activity. Collectively, our iBRN strategy provides the first compelling evidence to elucidate molecular aetiology in neurodegenerative diseases.
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http://dx.doi.org/10.1016/j.nbd.2021.105364DOI Listing
July 2021

Generation of a common marmoset embryonic stem cell line CMES40-OC harboring a POU5F1 (OCT4)-2A-mCerulean3 knock-in reporter allele.

Stem Cell Res 2021 05 24;53:102308. Epub 2021 Mar 24.

Department of Physiology, School of Medicine, Keio University, Tokyo, Japan; Laboratory for Marmoset Neural Architecture, RIKEN Center for Brain Science, Saitama, Japan.

POU class 5 homeobox 1 (POU5F1, also known as OCT4) is critical for maintenance of pluripotency, germ cell fate, reprogramming into a pluripotent state, and early embryogenesis. We generated an embryonic stem cell (ESC) line of the common marmoset (Callithrix jacchus) harboring a heterozygous knock-in allele of OCT4-P2A-mCerulean-T2A-pac. The ESC line (CMES40-OC) will be valuable for investigation of primed/naïve pluripotency and germ cell fate. Homozygous OCT4 knock-in clones were generated but could not be sustained in an undifferentiated state in long-term culture. The OCT4 knock-in system facilitated simultaneous knock-in of a reporter construct at another locus, DDX4 (VASA).
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http://dx.doi.org/10.1016/j.scr.2021.102308DOI Listing
May 2021

Non-viral Induction of Transgene-free iPSCs from Somatic Fibroblasts of Multiple Mammalian Species.

Stem Cell Reports 2021 Apr 1;16(4):754-770. Epub 2021 Apr 1.

Department of Physiology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, Japan; Laboratory for Marmoset Neural Architecture, RIKEN Center for Brain Science, Saitama, Japan. Electronic address:

Induced pluripotent stem cells (iPSCs) are capable of providing an unlimited source of cells from all three germ layers and germ cells. The derivation and usage of iPSCs from various animal models may facilitate stem cell-based therapy, gene-modified animal production, and evolutionary studies assessing interspecies differences. However, there is a lack of species-wide methods for deriving iPSCs, in particular by means of non-viral and non-transgene-integrating (NTI) approaches. Here, we demonstrate the iPSC derivation from somatic fibroblasts of multiple mammalian species from three different taxonomic orders, including the common marmoset (Callithrix jacchus) in Primates, the dog (Canis lupus familiaris) in Carnivora, and the pig (Sus scrofa) in Cetartiodactyla, by combinatorial usage of chemical compounds and NTI episomal vectors. Interestingly, the fibroblasts temporarily acquired a neural stem cell-like state during the reprogramming. Collectively, our method, robustly applicable to various species, holds a great potential for facilitating stem cell-based research using various animals in Mammalia.
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http://dx.doi.org/10.1016/j.stemcr.2021.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8072067PMC
April 2021

Establishment of an in vitro model for analyzing mitochondrial ultrastructure in PRKN-mutated patient iPSC-derived dopaminergic neurons.

Mol Brain 2021 03 23;14(1):58. Epub 2021 Mar 23.

Department of Cell Biology and Neuroscience, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.

Mitochondrial structural changes are associated with the regulation of mitochondrial function, apoptosis, and neurodegenerative diseases. PRKN is known to be involved with various mechanisms of mitochondrial quality control including mitochondrial structural changes. Parkinson's disease (PD) with PRKN mutations is characterized by the preferential degeneration of dopaminergic neurons in the substantia nigra pars compacta, which has been suggested to result from the accumulation of damaged mitochondria. However, ultrastructural changes of mitochondria specifically in dopaminergic neurons derived from iPSC have rarely been analyzed. The main reason for this would be that the dopaminergic neurons cannot be distinguished directly among a mixture of iPSC-derived differentiated cells under electron microscopy. To selectively label dopaminergic neurons and analyze mitochondrial morphology at the ultrastructural level, we generated control and PRKN-mutated patient tyrosine hydroxylase reporter (TH-GFP) induced pluripotent stem cell (iPSC) lines. Correlative light-electron microscopy analysis and live cell imaging of GFP-expressing dopaminergic neurons indicated that iPSC-derived dopaminergic neurons had smaller and less functional mitochondria than those in non-dopaminergic neurons. Furthermore, the formation of spheroid-shaped mitochondria, which was induced in control dopaminergic neurons by a mitochondrial uncoupler, was inhibited in the PRKN-mutated dopaminergic neurons. These results indicate that our established TH-GFP iPSC lines are useful for characterizing mitochondrial morphology, such as spheroid-shaped mitochondria, in dopaminergic neurons among a mixture of various cell types. Our in vitro model would provide insights into the vulnerability of dopaminergic neurons and the processes leading to the preferential loss of dopaminergic neurons in patients with PRKN mutations.
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http://dx.doi.org/10.1186/s13041-021-00771-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986497PMC
March 2021
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