論文 - 岡野　栄之

Large-area fluorescence and electron microscopic correlative imaging with multibeam scanning electron microscopy

Shibata S., Iseda T., Mitsuhashi T., Oka A., Shindo T., Moritoki N., Nagai T., Otsubo S., Inoue T., Sasaki E., Akazawa C., Takahashi T., Schalek R., Lichtman J., Okano H.

Frontiers in Neural Circuits （Frontiers in Neural Circuits）  13 2019年04月

ISSN  16625110

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© 2019 Shibata, Iseda, Mitsuhashi, Oka, Shindo, Moritoki, Nagai, Otsubo, Inoue, Sasaki, Akazawa, Takahashi, Schalek, Lichtman and Okano. Recent improvements in correlative light and electron microscopy (CLEM) technology have led to dramatic improvements in the ability to observe tissues and cells. Fluorescence labeling has been used to visualize the localization of molecules of interest through immunostaining or genetic modification strategies for the identification of the molecular signatures of biological specimens. Newer technologies such as tissue clearing have expanded the field of observation available for fluorescence labeling; however, the area of correlative observation available for electron microscopy (EM) remains restricted. In this study, we developed a large-area CLEM imaging procedure to show specific molecular localization in large-scale EM sections of mouse and marmoset brain. Target molecules were labeled with antibodies and sequentially visualized in cryostat sections using fluorescence and gold particles. Fluorescence images were obtained by light microscopy immediately after antibody staining. Immunostained sections were postfixed for EM, and silver-enhanced sections were dehydrated in a graded ethanol series and embedded in resin. Ultrathin sections for EM were prepared from fully polymerized resin blocks, collected on silicon wafers, and observed by multibeam scanning electron microscopy (SEM). Multibeam SEM has made rapid, large-area observation at high resolution possible, paving the way for the analysis of detailed structures using the CLEM approach. Here, we describe detailed methods for large-area CLEM in various tissues of both rodents and primates.

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Noninvasive technique to evaluate the muscle fiber characteristics using q-space imaging

Hata J., Nakashima D., Tsuji O., Fujiyoshi K., Yasutake K., Sera Y., Komaki Y., Hikishima K., Nagura T., Matsumoto M., Okano H., Nakamura M.

PLoS ONE （PLoS ONE）  14 （ 4 ）  2019年04月

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© 2019 Hata et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background Skeletal muscles include fast and slow muscle fibers. The tibialis anterior muscle (TA) is mainly composed of fast muscle fibers, whereas the soleus muscle (SOL) is mainly composed of slow muscle fibers. However, a noninvasive approach for appropriately investigating the characteristics of muscles is not available. Monitoring of skeletal muscle characteristics can help in the evaluation of the effects of strength training and diseases on skeletal muscles. Purpose The present study aimed to determine whether q-space imaging can distinguish between TA and SOL in in vivo mice. Methods In vivo magnetic resonance imaging of the right calves of mice (n = 8) was performed using a 7-Tesla magnetic resonance imaging system with a cryogenic probe. TA and SOL were assessed. q-space imaging was performed with a field of view of 10 mm × 10 mm, matrix of 48 × 48, and section thickness of 1000 μm. There were ten b-values ranging from 0 to 4244 s/mm 2 , and each b-value had diffusion encoding in three directions. Magnetic resonance imaging findings were compared with immunohistological findings. Results Full width at half maximum and Kurtosis maps of q-space imaging showed signal intensities consistent with immunohistological findings for both fast (myosin heavy chain II) and slow (myosin heavy chain I) muscle fibers. With regard to quantification, both full width at half maximum and Kurtosis could represent the immunohistological findings that the cell diameter of TA was larger than that of SOL (P < 0.01). Conclusion q-space imaging could clearly differentiate TA from SOL using differences in cell diameters. This technique is a promising method to noninvasively estimate the fiber type ratio in skeletal muscles, and it can be further developed as an indicator of muscle characteristics.

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Application of hepatocyte growth factor for acute spinal cord injury: The road from basic studies to human treatment

Kitamura K., Nagoshi N., Tsuji O., Matsumoto M., Okano H., Nakamura M.

International Journal of Molecular Sciences （International Journal of Molecular Sciences）  20 （ 5 ）  2019年03月

ISSN  16616596

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© 2019 by the authors. Licensee MDPI, Basel, Switzerland. Hepatocyte growth factor (HGF) was first identified as a potent mitogen for mature hepatocytes, and has also gained attention as a strong neurotrophic factor in the central nervous system. We found that during the acute phase of spinal cord injury (SCI) in rats, c-Met, the specific receptor for HGF, increases sharply, while the endogenous HGF up-regulation is relatively weak. Introducing exogenous HGF into the spinal cord by injecting an HGF-expressing viral vector significantly increased the neuron and oligodendrocyte survival, angiogenesis, and axonal regeneration, to reduce the area of damage and to promote functional recovery in rats after SCI. Other recent studies in rodents have shown that exogenously administered HGF during the acute phase of SCI reduces astrocyte activation to decrease glial scar formation, and exerts anti-inflammatory effects to reduce leukocyte infiltration. We also reported that the intrathecal infusion of recombinant human HGF (intrathecal rhHGF) improves neurological hand function after cervical contusive SCI in the common marmoset, a non-human primate. Based on these collective results, we conducted a phase I/II clinical trial of intrathecal rhHGF for patients with acute cervical SCI who showed a modified Frankel grade of A/B1/B2 72 h after injury onset, from June 2014 to May 2018.

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

Kojima K., Miyoshi H., Nagoshi N., Kohyama J., Itakura G., Kawabata S., Ozaki M., Iida T., Sugai K., Ito S., Fukuzawa R., Yasutake K., Renault-Mihara F., Shibata S., Matsumoto M., Nakamura M., Okano H.

Stem Cells Translational Medicine （Stem Cells Translational Medicine）  8 （ 3 ） 260 - 270 2019年03月

ISSN  21576564

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© 2018 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press Tumorigenesis is an important problem that needs to be addressed in the field of human stem/progenitor cell transplantation for the treatment of subacute spinal cord injury (SCI). When certain “tumorigenic” cell lines are transplanted into the spinal cord of SCI mice model, there is initial improvement of motor function, followed by abrupt deterioration secondary to the effect of tumor growth. A significant proportion of the transplanted cells remains undifferentiated after transplantation and is thought to increase the risk of tumorigenesis. In this study, using lentiviral vectors, we introduced the herpes simplex virus type 1 thymidine kinase (HSVtk) gene into a human induced pluripotent stem cell-derived neural stem/progenitor cell (hiPSC-NS/PC) line that is known to undergo tumorigenic transformation. Such approach enables selective ablation of the immature proliferating cells and thereby prevents subsequent tumor formation. In vitro, the HSVtk system successfully ablated the immature proliferative neural cells while preserving mature postmitotic neuronal cells. Similar results were observed in vivo following transplantation into the injured spinal cords of immune-deficient (nonobese diabetic–severe combined immune-deficient) mice. Ablation of the proliferating cells exerted a protective effect on the motor function which was regained after transplantation, simultaneously defending the spinal cord from the harmful tumor growth. These results suggest a potentially promising role of suicide genes in opposing tumorigenesis during stem cell therapy. This system allows both preventing and treating tumorigenesis following hiPSC-NS/PC transplantation without sacrificing the improved motor function. Stem Cells Translational Medicine 2019;8:260&270.

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Induced Pluripotent Stem Cells Reprogrammed with Three Inhibitors Show Accelerated Differentiation Potentials with High Levels of 2-Cell Stage Marker Expression

Nishihara K., Shiga T., Nakamura E., Akiyama T., Sasaki T., Suzuki S., Ko M., Tada N., Okano H., Akamatsu W.

Stem Cell Reports （Stem Cell Reports）  12 （ 2 ） 305 - 318 2019年02月

ISSN  22136711

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© 2019 The Authors Although pluripotent stem cells can generate various types of differentiated cells, it is unclear why lineage-committed stem/progenitor cells derived from pluripotent stem cells are decelerated and why the differentiation-resistant propensity of embryonic stem cell (ESC)/induced pluripotent stem cell (iPSC)-derived cells is predominant compared with the in vivo equivalents derived from embryonic/adult tissues. In this study, we demonstrated that iPSCs reprogrammed and maintained with three chemical inhibitors of the fibroblast growth factor 4-mitogen-activated protein kinase cascade and GSK3β (3i) could be differentiated into all three germ layers more efficiently than the iPSCs reprogrammed without the 3i chemicals, even though they were maintained with 3i chemicals once they were reprogrammed. Although the iPSCs reprogrammed with 3i had increased numbers of Zscan4-positive cells, the Zscan4-positive cells among iPSCs that were reprogrammed without 3i did not have an accelerated differentiation ability. These observations suggest that 3i exposure during the reprogramming period determines the accelerated differentiation/maturation potentials of iPSCs that are stably maintained at the distinct state.

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Increased cytotoxicity of herpes simplex virus thymidine kinase expression in human induced pluripotent stem cells

Iwasawa C., Tamura R., Sugiura Y., Suzuki S., Kuzumaki N., Narita M., Suematsu M., Nakamura M., Yoshida K., Toda M., Okano H., Miyoshi H.

International Journal of Molecular Sciences （International Journal of Molecular Sciences）  20 （ 4 ）  2019年02月

ISSN  16616596

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© 2019 by the authors. Licensee MDPI, Basel, Switzerland. Human induced pluripotent stem cells (iPSCs) hold enormous promise for regenerative medicine. The major safety concern is the tumorigenicity of transplanted cells derived from iPSCs. A potential solution would be to introduce a suicide gene into iPSCs as a safety switch. The herpes simplex virus type 1 thymidine kinase (HSV-TK) gene, in combination with ganciclovir, is the most widely used enzyme/prodrug suicide system from basic research to clinical applications. In the present study, we attempted to establish human iPSCs that stably expressed HSV-TK with either lentiviral vectors or CRISPR/Cas9-mediated genome editing. However, this task was difficult to achieve, because high-level and/or constitutive expression of HSV-TK resulted in the induction of cell death or silencing of HSV-TK expression. A nucleotide metabolism analysis suggested that excessive accumulation of thymidine triphosphate, caused by HSV-TK expression, resulted in an imbalance in the dNTP pools. This unbalanced state led to DNA synthesis inhibition and cell death in a process similar to a “thymidine block”, but more severe. We also demonstrated that the Tet-inducible system was a feasible solution for overcoming the cytotoxicity of HSV-TK expression. Our results provided a warning against using the HSV-TK gene in human iPSCs, particularly in clinical applications.

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Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Komatsu M., Kaneko T., Okano H., Ichinohe N.

Journal of visualized experiments : JoVE （Journal of visualized experiments : JoVE）   （ 144 ）  2019年02月

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Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

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A high-throughput neurohistological pipeline for brain-wide mesoscale connectivity mapping of the common marmoset

Lin M., Takahashi Y., Huo B., Hanada M., Nagashima J., Hata J., Tolpygo A., Ram K., Lee B., Miller M., Rosa M., Sasaki E., Iriki A., Okano H., Mitra P.

eLife （eLife）  8 2019年02月

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© Lin et al. Understanding the connectivity architecture of entire vertebrate brains is a fundamental but difficult task. Here we present an integrated neuro-histological pipeline as well as a grid-based tracer injection strategy for systematic mesoscale connectivity mapping in the common marmoset (Callithrix jacchus). Individual brains are sectioned into ~1700 20 mm sections using the tape transfer technique, permitting high quality 3D reconstruction of a series of histochemical stains (Nissl, myelin) interleaved with tracer labeled sections. Systematic in-vivo MRI of the individual animals facilitates injection placement into reference-atlas defined anatomical compartments. Further, by combining the resulting 3D volumes, containing informative cytoarchitectonic markers, with in-vivo and ex-vivo MRI, and using an integrated computational pipeline, we are able to accurately map individual brains into a common reference atlas despite the significant individual variation. This approach will facilitate the systematic assembly of a mesoscale connectivity matrix together with unprecedented 3D reconstructions of brain-wide projection patterns in a primate brain.

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Comparative principles for next-generation neuroscience

Miller C., Hale M., Okano H., Okabe S., Mitra P.

Frontiers in Behavioral Neuroscience （Frontiers in Behavioral Neuroscience）  13 2019年01月

ISSN  16625153

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© 2019 Miller, Hale, Okano, Okabe and Mitra. Neuroscience is enjoying a renaissance of discovery due in large part to the implementation of next-generation molecular technologies. The advent of genetically encoded tools has complemented existing methods and provided researchers the opportunity to examine the nervous system with unprecedented precision and to reveal facets of neural function at multiple scales. The weight of these discoveries, however, has been technique-driven from a small number of species amenable to the most advanced gene-editing technologies. To deepen interpretation and build on these breakthroughs, an understanding of nervous system evolution and diversity are critical. Evolutionary change integrates advantageous variants of features into lineages, but is also constrained by pre-existing organization and function. Ultimately, each species’ neural architecture comprises both properties that are species-specific and those that are retained and shared. Understanding the evolutionary history of a nervous system provides interpretive power when examining relationships between brain structure and function. The exceptional diversity of nervous systems and their unique or unusual features can also be leveraged to advance research by providing opportunities to ask new questions and interpret findings that are not accessible in individual species. As new genetic and molecular technologies are added to the experimental toolkits utilized in diverse taxa, the field is at a key juncture to revisit the significance of evolutionary and comparative approaches for next-generation neuroscience as a foundational framework for understanding fundamental principles of neural function.

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Reduced expression of somatostatin in GABAergic interneurons derived from induced pluripotent stem cells of patients with parkin mutations

Iwasawa C., Kuzumaki N., Suda Y., Kagawa R., Oka Y., Hattori N., Okano H., Narita M.

Molecular Brain （Molecular Brain）  12 （ 1 ）  2019年01月

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© 2019 The Author(s). Parkinson's disease (PD) is associated with both motor and non-motor symptoms, including constipation, sensory neuropathy, depression, dementia and sleep disorder. Somatostatin (SST) is considered to be a modulator of GABAergic inhibitory transmission, and its levels are reduced in cerebrospinal fluid of PD patients. In the present study, we evaluated the changes in the expression of SST in GABAergic neurons derived from induced pluripotent stem cells (iPSCs) of PD patients. Neural cells were co-treated with the Wnt antagonist IWP-2 and Shh during neurosphere formation to induce GABA-positive forebrain interneurons. Quantitative analyses showed no significant differences, but slight decreases, in the potency of differentiation into GABAergic neurons derived from iPSCs between healthy control and patients with PARK2 mutations, who have been classified as a type of early-onset familial PD due to mutations in the parkin gene. Under this condition, the mRNA level of SST in GABAergic interneurons derived from iPSCs of PARK2-specific PD patients significantly decreased as neural maturation progressed. We also found that SST-positive GABAergic neurons were clearly reduced in GABAergic neurons derived from iPSCs of patients with PARK2 mutations. These findings suggest that the reduction in the expression level of SST in GABAergic interneurons of PD may, at least partly, lead to complex PD-induced symptoms.

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Unidirectional monosynaptic connections from auditory areas to the primary visual cortex in the marmoset monkey

Majka P., Rosa M., Bai S., Chan J., Huo B., Jermakow N., Lin M., Takahashi Y., Wolkowicz I., Worthy K., Rajan R., Reser D., Wójcik D., Okano H., Mitra P.

Brain Structure and Function （Brain Structure and Function）  224 （ 1 ） 111 - 131 2019年01月

ISSN  18632653

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© 2018, The Author(s). Until the late twentieth century, it was believed that different sensory modalities were processed by largely independent pathways in the primate cortex, with cross-modal integration only occurring in specialized polysensory areas. This model was challenged by the finding that the peripheral representation of the primary visual cortex (V1) receives monosynaptic connections from areas of the auditory cortex in the macaque. However, auditory projections to V1 have not been reported in other primates. We investigated the existence of direct interconnections between V1 and auditory areas in the marmoset, a New World monkey. Labelled neurons in auditory cortex were observed following 4 out of 10 retrograde tracer injections involving V1. These projections to V1 originated in the caudal subdivisions of auditory cortex (primary auditory cortex, caudal belt and parabelt areas), and targeted parts of V1 that represent parafoveal and peripheral vision. Injections near the representation of the vertical meridian of the visual field labelled few or no cells in auditory cortex. We also placed 8 retrograde tracer injections involving core, belt and parabelt auditory areas, none of which revealed direct projections from V1. These results confirm the existence of a direct, nonreciprocal projection from auditory areas to V1 in a different primate species, which has evolved separately from the macaque for over 30 million years. The essential similarity of these observations between marmoset and macaque indicate that early-stage audiovisual integration is a shared characteristic of primate sensory processing.

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

Tsuji O., Sugai K., Yamaguchi R., Tashiro S., Nagoshi N., Kohyama J., Iida T., Ohkubo T., Itakura G., Isoda M., Shinozaki M., Fujiyoshi K., Kanemura Y., Yamanaka S., Nakamura M., Okano H.

Stem Cells （Stem Cells）  37 （ 1 ） 6 - 13 2019年01月

ISSN  10665099

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© 2018 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press There have been numerous attempts to develop stem cell transplantation approaches to promote the regeneration of spinal cord injury (SCI). Our multicenter team is currently planning to launch a first-in-human clinical study of an induced pluripotent stem cell (iPSC)-based cell transplant intervention for subacute SCI. This trial was conducted as class I regenerative medicine protocol as provided for under Japan's Act on the Safety of Regenerative Medicine, using neural stem/progenitor cells derived from a clinical-grade, integration-free human “iPSC stock” generated by the Kyoto University Center for iPS Cell Research and Application. In the present article, we describe how we are preparing to initiate this clinical study, including addressing the issues of safety and tumorigenesis as well as practical problems that must be overcome to enable the development of therapeutic interventions for patients with chronic SCI. Stem Cells 2019;37:6–13.

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PAT - Probabilistic axon tracking for densely labeled neurons in large 3-D micrographs

Skibbe H., Reisert M., Nakae K., Watakabe A., Hata J., Mizukami H., Okano H., Yamamori T., Ishii S.

IEEE Transactions on Medical Imaging （IEEE Transactions on Medical Imaging）  38 （ 1 ） 69 - 78 2019年01月

ISSN  02780062

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© 1982-2012 IEEE. A major goal of contemporary neuroscience research is to map the structural connectivity of mammalian brain using microscopy imaging data. In this context, the reconstruction of densely labeled axons from two-photon microscopy images is a challenging and important task. The visually overlapping, crossing, and often strongly distorted images of the axons allow many ambiguous interpretations to be made. We address the problem of tracking axons in densely labeled samples of neurons in large image data sets acquired from marmoset brains. Our high-resolution images were acquired using two-photon microscopy and they provided whole brain coverage, occupying terabytes of memory. Both the image distortions and the large data set size frequently make it impractical to apply present-day neuron tracing algorithms to such data due to the optimization of such algorithms to the precise tracing of either single or sparse sets of neurons. Thus, new tracking techniques are needed. We propose a probabilistic axon tracking algorithm (PAT). PAT tackles the tracking of axons in two steps: locally (L-PAT) and globally (G-PAT). L-PAT is a probabilistic tracking algorithm that can tackle distorted, cluttered images of densely labeled axons. L-PAT divides a large micrograph into smaller image stacks. It then processes each image stack independently before mapping the axons in each image to a sparse model of axon trajectories. G-PAT merges the sparse L-PAT models into a single global model of axon trajectories by minimizing a global objective function using a probabilistic optimization method. We demonstrate the superior performance of PAT over standard approaches on synthetic data. Furthermore, we successfully apply PAT to densely labeled axons in large images acquired from marmoset brains.

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Are We There Yet? How and When Specific Biotechnologies Will Improve Human Health

O'Day E., Hosta-Rigau L., Oyarzún D., Okano H., de Lorenzo V., von Kameke C., Alsafar H., Cao C., Chen G., Ji W., Roberts R., Ronaghi M., Yeung K., Zhang F., Lee S.

Biotechnology Journal （Biotechnology Journal）  14 （ 1 ）  2019年01月

ISSN  18606768

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© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Patient X: A 67-year-old Caucasian man slips on a patch of ice. He has abrasions to his hands and has sustained significant damage to his hip. At the emergency room, he informs clinicians he takes atorvastatin, metformin, and glimepiride to treat hypertension and Type 2 Diabetes Mellitus (T2DM). X-rays reveal a fractured hip, which will require total hip replacement surgery.

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Tau isoform expression and phosphorylation in marmoset brains

Sharma G., Huo A., Kimura T., Shiozawa S., Kobayashi R., Sahara N., Ishibashi M., Ishigaki S., Saito T., Ando K., Murayama S., Hasegawa M., Sobue G., Okano H., Hisanaga S.

Journal of Biological Chemistry （Journal of Biological Chemistry）  294 （ 30 ） 11433 - 11444 2019年

ISSN  00219258

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© 2019 Sharma et al. Tau is a microtubule-associated protein expressed in neuronal axons. Hyperphosphorylated tau is a major component of neurofibrillary tangles, a pathological hallmark of Alzheimer’s disease (AD). Hyperphosphorylated tau aggregates are also found in many neurodegenerative diseases, collectively referred to as “tauopathies,” and tau mutations are associated with familial frontotemporal lobar degeneration (FTLD). Previous studies have generated transgenic mice with mutant tau as tauopathy models, but nonhuman primates, which are more similar to humans, may be a better model to study tauopathies. For example, the common marmoset is poised as a nonhuman primate model for investigating the etiology of age-related neurodegenerative diseases. However, no biochemical studies of tau have been conducted in marmoset brains. Here, we investigated several important aspects of tau, including expression of different tau isoforms and its phosphorylation status, in the marmoset brain. We found that marmoset tau does not possess the “primate-unique motif” in its N-terminal domain. We also discovered that the tau isoform expression pattern in marmosets is more similar to that of mice than that of humans, with adult marmoset brains expressing only four-repeat tau isoforms as in adult mice but unlike in adult human brains. Of note, tau in brains of marmoset newborns was phosphorylated at several sites associated with AD pathology. However, in adult marmoset brains, much of this phosphorylation was lost, except for Ser-202 and Ser-404 phosphorylation. These results reveal key features of tau expression and phosphorylation in the marmoset brain, a potentially useful nonhuman primate model of neurodegenerative diseases.

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Relation of koniocellular layers of dorsal lateral geniculate to inferior pulvinar nuclei in common marmosets

Huo B., Zeater N., Lin M., Takahashi Y., Hanada M., Nagashima J., Lee B., Hata J., Zaheer A., Grünert U., Miller M., Rosa M., Okano H., Martin P., Mitra P.

European Journal of Neuroscience （European Journal of Neuroscience）  2019年

ISSN  0953816X

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© 2019 Federation of European Neuroscience Societies and John Wiley & Sons Ltd Traditionally, the dorsal lateral geniculate nucleus (LGN) and the inferior pulvinar (IPul) nucleus are considered as anatomically and functionally distinct thalamic nuclei. However, in several primate species it has also been established that the koniocellular (K) layers of LGN and parts of the IPul have a shared pattern of immunoreactivity for the calcium-binding protein calbindin. These calbindin-rich cells constitute a thalamic matrix system which is implicated in thalamocortical synchronisation. Further, the K layers and IPul are both involved in visual processing and have similar connections with retina and superior colliculus. Here, we confirmed the continuity between calbindin-rich cells in LGN K layers and the central lateral division of IPul (IPulCL) in marmoset monkeys. By employing a high-throughput neuronal tracing method, we found that both the K layers and IPulCL form comparable patterns of connections with striate and extrastriate cortices; these connections are largely different to those of the parvocellular and magnocellular laminae of LGN. Retrograde tracer-labelled cells and anterograde tracer-labelled axon terminals merged seamlessly from IPulCL into LGN K layers. These results support continuity between LGN K layers and IPulCL, providing an anatomical basis for functional congruity of this region of the dorsal thalamic matrix and calling into question the traditional segregation between LGN and the inferior pulvinar nucleus.

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In Utero Amniotic Fluid Stem Cell Therapy Protects Against Myelomeningocele via Spinal Cord Coverage and Hepatocyte Growth Factor Secretion

Abe Y., Ochiai D., Masuda H., Sato Y., Otani T., Fukutake M., Ikenoue S., Miyakoshi K., Okano H., Tanaka M.

Stem Cells Translational Medicine （Stem Cells Translational Medicine）  2019年

ISSN  21576564

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© 2019 The Authors. Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press Despite the poor prognosis associated with myelomeningocele (MMC), the options for prenatal treatments are still limited. Recently, fetal cellular therapy has become a new option for treating birth defects, although the therapeutic effects and mechanisms associated with such treatments remain unclear. The use of human amniotic fluid stem cells (hAFSCs) is ideal with respect to immunoreactivity and cell propagation. The prenatal diagnosis of MMC during early stages of pregnancy could allow for the ex vivo proliferation and modulation of autologous hAFSCs for use in utero stem cell therapy. Therefore, we investigated the therapeutic effects and mechanisms of hAFSCs-based treatment for fetal MMC. hAFSCs were isolated as CD117-positive cells from the amniotic fluid of 15- to 17-week pregnant women who underwent amniocentesis for prenatal diagnosis and consented to this study. Rat dams were exposed to retinoic acid to induce fetal MMC and were subsequently injected with hAFSCs in each amniotic cavity. We measured the exposed area of the spinal cord and hepatocyte growth factor (HGF) levels at the lesion. The exposed spinal area of the hAFSC-treated group was significantly smaller than that of the control group. Immunohistochemical analysis demonstrated a reduction in neuronal damage such as neurodegeneration and astrogliosis in the hAFSC-treated group. Additionally, in lesions of the hAFSC-treated group, HGF expression was upregulated and HGF-positive hAFSCs were identified, suggesting that these cells migrated to the lesion and secreted HGF to suppress neuronal damage and induce neurogenesis. Therefore, in utero hAFSC therapy could become a novel strategy for fetal MMC. Stem Cells Translational Medicine 2019.

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Comparison of inner ear drug availability of combined treatment with systemic or local drug injections alone

Li Y., Kanzaki S., Shibata S., Nakamura M., Ozaki M., Okano H., Ogawa K.

Neuroscience Research （Neuroscience Research）  2019年

ISSN  01680102

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© 2019 Combination of systemic and local drug therapy has been proved more effective and safer for idiopathic sudden sensorineural hearing loss by some clinical trials, and there are few laboratory researches on its pharmacokinetic behaviors in the inner ear. In the present study, we use a new in vivo imaging system to compare the pharmacokinetics of combined therapy (CT), with intravenous (IV) or transtympanic (TT) injection alone in both ears of transgenic GFAP-Luc mice. Biological half-life, total photon counts and the area under the curve (AUC) value significantly increased after CT. However, adding IV to TT injection cannot strengthen the peak photon of the drug in the inner ear. In addition, when D-luciferin is injected to the left ear the volume of total photon count and AUC value of CT-left ear are larger than the combined volume of TT-left ear and IV-left ear, suggesting a synergistic effect, and those of CT-right ear are almost equal to the summation of those of IV-right group and TT-right group, suggesting no amplifying effect on the risk of systemic side effect. This study showed that CT could deliver more drugs into the inner ear, and brought a longer therapeutic window, and were more effective than intravenous or transtympanic injection alone in the pharmacokinetics.

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A versatile toolbox for knock-in gene targeting based on the Multisite Gateway technology

Yoshimatsu S., Sone T., Nakajima M., Sato T., Okochi R., Ishikawa M., Nakamura M., Sasaki E., Shiozawa S., Okano H.

PLoS ONE （PLoS ONE）  14 （ 8 ）  2019年

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© 2019 Yoshimatsu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Knock-in (KI) gene targeting can be employed for a wide range of applications in stem cell research. However, vectors for KI require multiple complicated processes for construction, including multiple times of digestion/ligation steps and extensive restriction mapping, which has imposed limitations for the robust applicability of KI gene targeting. To circumvent this issue, here we introduce versatile and systematic methods for generating KI vectors by molecular cloning. In this approach, we employed the Multisite Gateway technology, an efficient in vitro DNA recombination system using proprietary sequences and enzymes. KI vector construction exploiting these methods requires only efficient steps, such as PCR and recombination, enabling robust KI gene targeting. We show that combinatorial usage of the KI vectors generated using this method and site-specific nucleases enabled the precise integration of fluorescent protein genes in multiple loci of human and common marmoset (marmoset; Callithrix jacchus) pluripotent stem cells. The methods described here will facilitate the usage of KI technology and ultimately help to accelerate stem cell research.

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

Okubo T., Nagoshi N., Kohyama J., Tsuji O., Shinozaki M., Shibata S., Kase Y., Matsumoto M., Nakamura M., Okano H.

Stem Cell Reports （Stem Cell Reports）  11 （ 6 ） 1416 - 1432 2018年12月

ISSN  22136711

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© 2018 The Authors In this article, Okano and colleagues show that the GSI-treated hiPSC-NS/PCs caused significantly enhanced axonal regrowth, remyelination, inhibitory synapse formation with the host neural circuitry, and reticulo spinal tract fiber formation. These favorable outcomes contributed to motor function improvement. Therefore, treating cells with GSI provides a beneficial effect after transplantation, even in the chronic phase following SCI.

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