研究者業績

魚崎 英毅

Hideki Uosaki

基本情報

所属
自治医科大学 医学部 生化学講座 機能生化学部門 教授

J-GLOBAL ID
201301025555991898
researchmap会員ID
B000227412

外部リンク

Research interests: Heart Development, Stem Cells (ES/iPS cells)

Google Scholar - My Citation
http://scholar.google.com/citations?hl=en&user=F_3DQqUAAAAJ


学歴

 2

論文

 46
  • 魚崎英毅, CHANTHRA Nawin
    カレントテラピー 42(1) 39-44 2024年1月  招待有り筆頭著者責任著者
  • 徳山 剛士, 魚崎 英毅
    生体の科学 74(5) 396-397 2023年10月15日  招待有り最終著者責任著者
  • Tomohiro Hino, Satoshi N Omura, Ryoya Nakagawa, Tomoki Togashi, Satoru N Takeda, Takafumi Hiramoto, Satoshi Tasaka, Hisato Hirano, Takeshi Tokuyama, Hideki Uosaki, Soh Ishiguro, Madina Kagieva, Hiroyuki Yamano, Yuki Ozaki, Daisuke Motooka, Hideto Mori, Yuhei Kirita, Yoshiaki Kise, Yuzuru Itoh, Satoaki Matoba, Hiroyuki Aburatani, Nozomu Yachie, Tautvydas Karvelis, Virginijus Siksnys, Tsukasa Ohmori, Atsushi Hoshino, Osamu Nureki
    Cell 186(22) 4920-4935 2023年9月29日  査読有り
    SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) vectors. The type V-F Cas12f from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been harnessed as a compact genome-editing tool. Here, we developed an approach, combining deep mutational scanning and structure-informed design, to successfully generate two AsCas12f activity-enhanced (enAsCas12f) variants. Remarkably, the enAsCas12f variants exhibited genome-editing activities in human cells comparable with those of SpCas9 and AsCas12a. The cryoelectron microscopy (cryo-EM) structures revealed that the mutations stabilize the dimer formation and reinforce interactions with nucleic acids to enhance their DNA cleavage activities. Moreover, enAsCas12f packaged with partner genes in an all-in-one AAV vector exhibited efficient knock-in/knock-out activities and transcriptional activation in mice. Taken together, enAsCas12f variants could offer a minimal genome-editing platform for in vivo gene therapy.
  • Takafumi Hiramoto, Yuji Kashiwakura, Morisada Hayakawa, Nemekhbayar Baatartsogt, Nobuhiko Kamoshita, Tomoyuki Abe, Hiroshi Inaba, Hiroshi Nishimasu, Hideki Uosaki, Yutaka Hanazono, Osamu Nureki, Tsukasa Ohmori
    Communications Medicine 3(1) 2023年4月19日  査読有り
    Abstract Background Base editing via CRISPR-Cas9 has garnered attention as a method for correcting disease-specific mutations without causing double-strand breaks, thereby avoiding large deletions and translocations in the host chromosome. However, its reliance on the protospacer adjacent motif (PAM) can limit its use. We aimed to restore a disease mutation in a patient with severe hemophilia B using base editing with SpCas9-NG, a modified Cas9 with the board PAM flexibility. Methods We generated induced pluripotent stem cells (iPSCs) from a patient with hemophilia B (c.947T>C; I316T) and established HEK293 cells and knock-in mice expressing the patient’s F9 cDNA. We transduced the cytidine base editor (C>T), including the nickase version of Cas9 (wild-type SpCas9 or SpCas9-NG), into the HEK293 cells and knock-in mice through plasmid transfection and an adeno-associated virus vector, respectively. Results Here we demonstrate the broad PAM flexibility of SpCas9-NG near the mutation site. The base-editing approach using SpCas9-NG but not wild-type SpCas9 successfully converts C to T at the mutation in the iPSCs. Gene-corrected iPSCs differentiate into hepatocyte-like cells in vitro and express substantial levels of F9 mRNA after subrenal capsule transplantation into immunodeficient mice. Additionally, SpCas9-NG–mediated base editing corrects the mutation in both HEK293 cells and knock-in mice, thereby restoring the production of the coagulation factor. Conclusion A base-editing approach utilizing the broad PAM flexibility of SpCas9-NG can provide a solution for the treatment of genetic diseases, including hemophilia B.
  • 徳山剛士, 魚崎英毅
    実験医学 41(5) 720-726 2023年3月  招待有り最終著者責任著者
  • Chika Watanabe, Hitoshi Osaka, Miyuki Watanabe, Akihiko Miyauchi, Eriko F Jimbo, Takeshi Tokuyama, Hideki Uosaki, Yoshihito Kishita, Yasushi Okazaki, Takanori Onuki, Tomohiro Ebihara, Kenichi Aizawa, Kei Murayama, Akira Ohtake, Takanori Yamagata
    Molecular genetics and metabolism reports 34 100951-100951 2023年3月  査読有り
    Coenzyme Q10 (CoQ10) is involved in ATP production through electron transfer in the mitochondrial respiratory chain complex. CoQ10 receives electrons from respiratory chain complex I and II to become the reduced form, and then transfers electrons at complex III to become the oxidized form. The redox state of CoQ10 has been reported to be a marker of the mitochondrial metabolic state, but to our knowledge, no reports have focused on the individual quantification of reduced and oxidized CoQ10 or the ratio of reduced to total CoQ10 (reduced/total CoQ10) in patients with mitochondrial diseases. We measured reduced and oxidized CoQ10 in skin fibroblasts from 24 mitochondrial disease patients, including 5 primary CoQ10 deficiency patients and 10 respiratory chain complex deficiency patients, and determined the reduced/total CoQ10 ratio. In primary CoQ10 deficiency patients, total CoQ10 levels were significantly decreased, however, the reduced/total CoQ10 ratio was not changed. On the other hand, in mitochondrial disease patients other than primary CoQ10 deficiency patients, total CoQ10 levels did not decrease. However, the reduced/total CoQ10 ratio in patients with respiratory chain complex IV and V deficiency was higher in comparison to those with respiratory chain complex I deficiency. Measurement of CoQ10 in fibroblasts proved useful for the diagnosis of primary CoQ10 deficiency. In addition, the reduced/total CoQ10 ratio may reflect the metabolic status of mitochondrial disease.
  • Matthew Miyamoto, Suraj Kannan, Matthew J Anderson, Xihe Liu, David Suh, Myo Htet, Biyi Li, Tejasvi Kakani, Sean Murphy, Emmanouil Tampakakis, Mark Lewandoski, Peter Andersen, Hideki Uosaki, Chulan Kwon
    Proceedings of the National Academy of Sciences of the United States of America 120(4) e2217687120 2023年1月24日  査読有り
    The heart develops in a synchronized sequence of proliferation and differentiation of cardiac progenitor cells (CPCs) from two anatomically distinct pools of cells, the first heart field (FHF) and second heart field (SHF). Congenital heart defects arise upon dysregulation of these processes, many of which are restricted to derivatives of the FHF or SHF. Of the conserved set of signaling pathways that regulate development, the Wnt signaling pathway has long been known for its importance in SHF development. The source of such Wnts has remained elusive, though it has been postulated that these Wnts are secreted from ectodermal or endodermal sources. The central question remains unanswered: Where do these Wnts come from? Here, we show that CPCs autoregulate SHF development via Wnt through genetic manipulation of a key Wnt export protein (Wls), scRNA-seq analysis of CPCs, and use of our precardiac organoid system. Through this, we identify dysregulated developmental trajectories of anterior SHF cell fate, leading to a striking single ventricle phenotype in knockout embryos. We then applied our findings to our precardiac organoid model and found that Wnt2 is sufficient to restore SHF cell fate in our model of disrupted endogenous Wnt signaling. In this study, we provide a basis for SHF cell fate decision-proliferation vs. differentiation-autoregulated by CPCs through Wnt.
  • Razan E Ahmed, Takeshi Tokuyama, Tatsuya Anzai, Nawin Chanthra, Hideki Uosaki
    Philosophical transactions of the Royal Society of London. Series B, Biological sciences 377(1864) 20210325-20210325 2022年11月21日  査読有り招待有り最終著者責任著者
    During postnatal cardiac development, cardiomyocytes mature and turn into adult ones. Hence, all cellular properties, including morphology, structure, physiology and metabolism, are changed. One of the most important aspects is the contractile apparatus, of which the minimum unit is known as a sarcomere. Sarcomere maturation is evident by enhanced sarcomere alignment, ultrastructural organization and myofibrillar isoform switching. Any maturation process failure may result in cardiomyopathy. Sarcomere function is intricately related to other organelles, and the growing evidence suggests reciprocal regulation of sarcomere and mitochondria on their maturation. Herein, we summarize the molecular mechanism that regulates sarcomere maturation and the interplay between sarcomere and other organelles in cardiomyocyte maturation. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.
  • Sandeep Kambhampati, Sean Murphy, Hideki Uosaki, Chulan Kwon
    Journal of Computational Biology 29(9) 1031-1044 2022年9月5日  責任著者
  • Takeshi Tokuyama, Hideki Uosaki, Ayumu Sugiura, Gen Nishitai, Keisuke Takeda, Shun Nagashima, Isshin Shiiba, Naoki Ito, Taku Amo, Satoshi Mohri, Akiyuki Nishimura, Motohiro Nishida, Ayumu Konno, Hirokazu Hirai, Satoshi Ishido, Takahiro Yoshizawa, Takayuki Shindo, Shingo Takada, Shintaro Kinugawa, Ryoko Inatome, Shigeru Yanagi
    iScience 25(7) 104582-104582 2022年7月15日  査読有り
    Abnormal mitochondrial fragmentation by dynamin-related protein1 (Drp1) is associated with the progression of aging-associated heart diseases, including heart failure and myocardial infarction (MI). Here, we report a protective role of outer mitochondrial membrane (OMM)-localized E3 ubiquitin ligase MITOL/MARCH5 against cardiac senescence and MI, partly through Drp1 clearance by OMM-associated degradation (OMMAD). Persistent Drp1 accumulation in cardiomyocyte-specific MITOL conditional-knockout mice induced mitochondrial fragmentation and dysfunction, including reduced ATP production and increased ROS generation, ultimately leading to myocardial senescence and chronic heart failure. Furthermore, ischemic stress-induced acute downregulation of MITOL, which permitted mitochondrial accumulation of Drp1, resulted in mitochondrial fragmentation. Adeno-associated virus-mediated delivery of the MITOL gene to cardiomyocytes ameliorated cardiac dysfunction induced by MI. Our findings suggest that OMMAD activation by MITOL can be a therapeutic target for aging-associated heart diseases, including heart failure and MI.
  • Chihiro Nishiyama, Yuichi Saito, Akane Sakaguchi, Mari Kaneko, Hiroshi Kiyonari, Yuqing Xu, Yuichiro Arima, Hideki Uosaki, Wataru Kimura
    Circulation 146(2) 101161CIRCULATIONAHA121055269-139 2022年5月26日  査読有り
    BACKGROUND: Early neonates of both large and small mammals are able to regenerate the myocardium through cardiomyocyte proliferation for only a short period after birth. This myocardial regenerative capacity declines in parallel with withdrawal of cardiomyocytes from the cell cycle in the first few postnatal days. No mammalian species examined to date has been found capable of a meaningful regenerative response to myocardial injury later than 1 week after birth. METHODS: We examined cardiomyocyte proliferation in neonates of the marsupial opossum (Monodelphis domestica) by immunostaining at various times after birth. The regenerative capacity of the postnatal opossum myocardium was assessed after either apex resection or induction of myocardial infarction at postnatal day 14 or 29, whereas that of the postnatal mouse myocardium was assessed after myocardial infarction at postnatal day 7. Bioinformatics data analysis, immunofluorescence staining, and pharmacological and genetic intervention were applied to determine the role of AMPK (5'-AMP-activated protein kinase) signaling in regulation of the mammalian cardiomyocyte cell cycle. RESULTS: Opossum neonates were found to manifest cardiomyocyte proliferation for at least 2 weeks after birth at a frequency similar to that apparent in early neonatal mice. Moreover, the opossum heart at postnatal day 14 showed substantial regenerative capacity both after apex resection and after myocardial infarction injury, whereas this capacity had diminished by postnatal day 29. Transcriptomic and immunofluorescence analyses indicated that AMPK signaling is activated in postnatal cardiomyocytes of both opossum and mouse. Pharmacological or genetic inhibition of AMPK signaling was sufficient to extend the postnatal window of cardiomyocyte proliferation in both mouse and opossum neonates as well as of cardiac regeneration in neonatal mice. CONCLUSIONS: The marsupial opossum maintains cardiomyocyte proliferation and a capacity for myocardial regeneration for at least 2 weeks after birth. As far as we are aware, this is the longest postnatal duration of such a capacity among mammals examined to date. AMPK signaling was implicated as an evolutionarily conserved regulator of mammalian postnatal cardiomyocyte proliferation.
  • 徳山剛士, 安済達也, 魚崎英毅
    小児内科 54(4) 635-639 2022年4月  招待有り最終著者責任著者
  • William J. Kowalski, Iris H. Garcia-Pak, Wenling Li, Hideki Uosaki, Emmanouil Tampakakis, Jizhong Zou, Yongshun Lin, Kira Patterson, Chulan Kwon, Yoh-Suke Mukouyama
    Frontiers in Cell and Developmental Biology 10 2022年3月11日  査読有り
    <jats:p>Embryos devoid of autonomic innervation suffer sudden cardiac death. However, whether autonomic neurons have a role in heart development is poorly understood. To investigate if sympathetic neurons impact cardiomyocyte maturation, we co-cultured phenotypically immature cardiomyocytes derived from human induced pluripotent stem cells with mouse sympathetic ganglion neurons. We found that 1) multiple cardiac structure and ion channel genes related to cardiomyocyte maturation were up-regulated when co-cultured with sympathetic neurons; 2) sarcomere organization and connexin-43 gap junctions increased; 3) calcium imaging showed greater transient amplitudes. However, sarcomere spacing, relaxation time, and level of sarcoplasmic reticulum calcium did not show matured phenotypes. We further found that addition of endothelial and epicardial support cells did not enhance maturation to a greater extent beyond sympathetic neurons, while administration of isoproterenol alone was insufficient to induce changes in gene expression. These results demonstrate that sympathetic neurons have a significant and complex role in regulating cardiomyocyte development.</jats:p>
  • Matthew Miyamoto, Peter Andersen, Edrick Sulistio, Xihe Liu, Sean Murphy, Suraj Kannan, Lucy Nam, William Miyamoto, Emmanouil Tampakakis, Narutoshi Hibino, Hideki Uosaki, Chulan Kwon
    Biochemical and Biophysical Research Communications 577 12-16 2021年11月5日  査読有り
  • Takeshi Tokuyama, Razan Elfadil Ahmed, Nawin Chanthra, Tatsuya Anzai, Hideki Uosaki
    Biology 10(10) 981-981 2021年9月29日  最終著者責任著者
    Mitochondrial cardiomyopathy (MCM) is characterized as an oxidative phosphorylation disorder of the heart. More than 100 genetic variants in nuclear or mitochondrial DNA have been associated with MCM. However, the underlying molecular mechanisms linking genetic variants to MCM are not fully understood due to the lack of appropriate cellular and animal models. Patient-specific induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) provide an attractive experimental platform for modeling cardiovascular diseases and predicting drug efficacy to such diseases. Here we introduce the pathological and therapeutic studies of MCM using iPSC-CMs and discuss the questions and latest strategies for research using iPSC-CMs.
  • Takafumi Miyamoto, Hideki Uosaki, Yuhei Mizunoe, Song-Iee Han, Satoi Goto, Daisuke Yamanaka, Masato Masuda, Yosuke Yoneyama, Hideki Nakamura, Naoko Hattori, Yoshinori Takeuchi, Hiroshi Ohno, Motohiro Sekiya, Takashi Matsuzaka, Fumihiko Hakuno, Shin-Ichiro Takahashi, Naoya Yahagi, Koichi Ito, Hitoshi Shimano
    Cell Reports Methods 1(4) 100052-100052 2021年8月23日  査読有り
  • Tatsuya Anzai, Hiromasa Hara, Nawin Chanthra, Taketaro Sadahiro, Masaki Ieda, Yutaka Hanazono, Hideki Uosaki
    Methods in molecular biology (Clifton, N.J.) 2320 247-259 2021年7月  招待有り最終著者
    A knock-in can generate fluorescent or Cre-reporter under the control of an endogenous promoter. It also generates knock-out or tagged-protein with fluorescent protein and short tags for tracking and purification. Recent advances in genome editing with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) significantly increased the efficiencies of making knock-in cells. Here we describe the detailed protocols of generating knock-in mouse and human pluripotent stem cells (PSCs) by electroporation and lipofection, respectively.
  • Azumi Noguchi, Kenji Ito, Yuichi Uosaki, Maky Ideta-Otsuka, Katsuhide Igarashi, Hideyuki Nakashima, Toshikazu Kakizaki, Ruri Kaneda, Hideki Uosaki, Yuchio Yanagawa, Kinichi Nakashima, Hirokazu Arakawa, Takumi Takizawa
    Neuroscience Research 173 22-33 2021年5月  査読有り
    Gene expression programs and concomitant chromatin regulation change dramatically during the maturation of postmitotic neurons. Subnuclear positioning of gene loci is relevant to transcriptional regulation. However, little is known about subnuclear genome positioning in neuronal maturation. Using cultured murine hippocampal neurons, we found genomic locus 14qD2 to be enriched with genes that are upregulated during neuronal maturation. Reportedly, the locus is homologous to human 8p21.3, which has been extensively studied in neuropsychiatry and neurodegenerative diseases. Mapping of the 14qD2 locus in the nucleus revealed that it was relocated from the nuclear periphery to the interior. Moreover, we found a concomitant decrease in lamin B1 expression. Overexpression of lamin B1 in neurons using a lentiviral vector prevented the relocation of the 14qD2 locus and repressed the transcription of the Egr3 gene on this locus. Taken together, our results suggest that reduced lamin B1 expression during the maturation of neurons is important for appropriate subnuclear positioning of the genome and transcriptional programs.
  • Suvd Byambaa, Hideki Uosaki, Tsukasa Ohmori, Hiromasa Hara, Hitoshi Endo, Osamu Nureki, Yutaka Hanazono
    Molecular therapy. Methods & clinical development 20 451-462 2021年3月12日  査読有り
    We conducted two lines of genome-editing experiments of mouse hematopoietic stem cells (HSCs) with the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9). First, to evaluate the genome-editing efficiency in mouse bona fide HSCs, we knocked out integrin alpha 2b (Itga2b) with Cas9 ribonucleoprotein (Cas9/RNP) and performed serial transplantation in mice. The knockout efficiency was estimated at approximately 15%. Second, giving an example of X-linked severe combined immunodeficiency (X-SCID) as a target genetic disease, we showed a proof-of-concept of universal gene correction, allowing rescue of most of X-SCID mutations, in a completely non-viral setting. We inserted partial cDNA of interleukin-2 receptor gamma chain (Il2rg) into intron 1 of Il2rg via non-homologous end-joining (NHEJ) with Cas9/RNP and a homology-independent targeted integration (HITI)-based construct. Repaired HSCs reconstituted T lymphocytes and thymuses in SCID mice. Our results show that a non-viral genome-editing of HSCs with CRISPR/Cas9 will help cure genetic diseases.
  • Sean A Murphy, Matthew Miyamoto, Anaïs Kervadec, Suraj Kannan, Emmanouil Tampakakis, Sandeep Kambhampati, Brian Leei Lin, Sam Paek, Peter Andersen, Dong-Ik Lee, Renjun Zhu, Steven S An, David A Kass, Hideki Uosaki, Alexandre R Colas, Chulan Kwon
    Nature communications 12(1) 1648-1648 2021年3月12日  査読有り
    Cardiomyocytes undergo significant structural and functional changes after birth, and these fundamental processes are essential for the heart to pump blood to the growing body. However, due to the challenges of isolating single postnatal/adult myocytes, how individual newborn cardiomyocytes acquire multiple aspects of the mature phenotype remains poorly understood. Here we implement large-particle sorting and analyze single myocytes from neonatal to adult hearts. Early myocytes exhibit wide-ranging transcriptomic and size heterogeneity that is maintained until adulthood with a continuous transcriptomic shift. Gene regulatory network analysis followed by mosaic gene deletion reveals that peroxisome proliferator-activated receptor coactivator-1 signaling, which is active in vivo but inactive in pluripotent stem cell-derived cardiomyocytes, mediates the shift. This signaling simultaneously regulates key aspects of cardiomyocyte maturation through previously unrecognized proteins, including YAP1 and SF3B2. Our study provides a single-cell roadmap of heterogeneous transitions coupled to cellular features and identifies a multifaceted regulator controlling cardiomyocyte maturation.
  • Razan E. Ahmed, Nawin Chanthra, Tatsuya Anzai, Keiichiro Koiwai, Tomoki Murakami, Hiroaki Suzuki, Yutaka Hanazono, Hideki Uosaki
    Journal of Visualized Experiments (169) 2021年3月3日  査読有り最終著者責任著者
    Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells provide promising sources for cardiac disease modeling. For cardiomyopathies, sarcomere shortening is one of the standard physiological assessments that are used with adult cardiomyocytes to examine their disease phenotypes. However, the available methods are not appropriate to assess the contractility of PSC-CMs, as these cells have underdeveloped sarcomeres that are invisible under phase-contrast microscopy. To address this issue and to perform sarcomere shortening with PSC-CMs, fluorescent-tagged sarcomere proteins and fluorescent live-imaging were used. Thin Z-lines and an M-line reside at both ends and the center of a sarcomere, respectively. Z-line proteins - α-Actinin (ACTN2), Telethonin (TCAP), and actin-associated LIM protein (PDLIM3) - and one M-line protein - Myomesin-2 (Myom2) - were tagged with fluorescent proteins. These tagged proteins can be expressed from endogenous alleles as knock-ins or from adeno-associated viruses (AAVs). Here, we introduce the methods to differentiate mouse and human pluripotent stem cells to cardiomyocytes, to produce AAVs, and to perform and analyze live-imaging. We also describe the methods for producing polydimethylsiloxane (PDMS) stamps for a patterned culture of PSC-CMs, which facilitates the analysis of sarcomere shortening with fluorescent-tagged proteins. To assess sarcomere shortening, time-lapse images of the beating cells were recorded at a high framerate (50-100 frames per second) under electrical stimulation (0.5-1 Hz). To analyze sarcomere length over the course of cell contraction, the recorded time-lapse images were subjected to SarcOptiM, a plug-in for ImageJ/Fiji. Our strategy provides a simple platform for investigating cardiac disease phenotypes in PSC-CMs.
  • Tomoyuki Abe, Hideki Uosaki, Hiroaki Shibata, Hiromasa Hara, Borjigin Sarentonglaga, Yoshikazu Nagao, Yutaka Hanazono
    Experimental Hematology 2021年3月  査読有り
  • Suvd Byambaa, Hideki Uosaki, Hiromasa Hara, Yasumitsu Nagao, Tomoyuki Abe, Hiroaki Shibata, Osamu Nureki, Tsukasa Ohmori, Yutaka Hanazono
    Experimental animals 69(2) 189-198 2020年4月24日  査読有り
    X-linked severe combined immunodeficiency (X-SCID) is an inherited genetic disorder. A majority of X-SCID subjects carries point mutations in the Interleukin-2 receptor gamma chain (IL2RG) gene. In contrast, Il2rg-knockout mice recapitulating X-SCID phenotype lack a large part of Il2rg instead of point mutations. In this study, we generated novel X-SCID mouse strains with small insertion and deletion (InDel) mutations in Il2rg by using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. To this end, we injected Streptococcus pyogenes Cas9 (SpCas9) mRNA and single guide RNA targeting the exon 2, 3 or 4 of Il2rg into mouse zygotes. In the F0 generation, we obtained 35 pups and 25 out of them were positive for Surveyor assay, and most of mutants displayed dramatic reductions of T and B lymphocytes in the peripheral blood. By amplicon sequencing, 15 out of 31 founder mice were determined as monoallelic mutants with possible minor mosaicisms while 10 mice were mosaic. Finally, we established new strains with 7-nucleotide deletion and 1-nucleotide insertions in the exon 2 and the exons 3 and 4, respectively. Although no IL2RG protein was detected on T cells of exons 3 and 4 mutants, IL2RG protein was unexpectedly detected in the exon 2 mutants. These data indicated that CRISPR/Cas9 targeting Il2rg causes InDel mutations effectively and generates genetically X-SCID mice. Genetic mutations, however, did not necessarily grant phenotypical alteration, which requires an intensive analysis after establishing a strain to confirm their phenotypes.
  • Tatsuya Anzai, Takanori Yamagata, Hideki Uosaki
    Frontiers in cell and developmental biology 8 268-268 2020年4月22日  査読有り最終著者責任著者
    Transcriptome landscape of organs from mice and humans offers perspectives on the process of how organs develop and the similarity and diversity in each organ between the species. Among multi-species and multi-organ dataset, which was previously generated, we focused on the mouse and human dataset and performed a reanalysis to provide a more specific perspective on the maturation of human cardiomyocytes. First, we examined how organs diversify their transcriptome during development across and within two species. We unexpectedly identified that ribosomal genes were differentially expressed between mice and humans. Second, we examined the corresponding ages of organs in mice and humans and found that the corresponding developmental ages did not match throughout organs. Mouse hearts at P0-3 and human hearts at 18-19 wpc showed the most proximity in the regard of the transcriptome. Third, we identified a novel set of maturation marker genes that are more consistent between mice and humans. In contrast, conventionally used maturation marker genes only work well with mouse hearts. Finally, we compared human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) in maturation-enhanced conditions to human fetal and adult hearts and revealed that human PSC-CMs only expressed low levels of the potential maturation marker genes. Our findings provide a novel foundation to study cardiomyocyte maturation and highlight the importance of studying human samples rather than relying on a mouse time-series dataset.
  • Hirofumi Nishizono, Mohamed Darwish, Hideki Uosaki, Nanami Masuyama, Motoaki Seki, Hiroyuki Abe, Nozomu Yachie, Ryohei Yasuda
    Journal of visualized experiments : JoVE (158) 2020年4月2日  査読有り
    The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.
  • Razan Elfadil Ahmed, Tatsuya Anzai, Nawin Chanthra, Hideki Uosaki
    Frontiers in cell and developmental biology 8 178-178 2020年3月19日  査読有り最終著者責任著者
    Cardiovascular diseases are the leading cause of death worldwide. Therefore, the discovery of induced pluripotent stem cells (iPSCs) and the subsequent generation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) was a pivotal point in regenerative medicine and cardiovascular research. They constituted an appealing tool for replacing dead and dysfunctional cardiac tissue, screening cardiac drugs and toxins, and studying inherited cardiac diseases. The problem is that these cells remain largely immature, and in order to utilize them, they must reach a functional degree of maturity. To attempt to mimic in vivo environment, various methods including prolonging culture time, co-culture and modulations of chemical, electrical, mechanical culture conditions have been tried. In addition to that, changing the topology of the culture made huge progress with the introduction of the 3D culture that closely resembles the in vivo cardiac topology and overcomes many of the limitations of the conventionally used 2D models. Nonetheless, 3D culture alone is not enough, and using a combination of these methods is being explored. In this review, we summarize the main differences between immature, fetal-like hiPSC-CMs and adult cardiomyocytes, then glance at the current approaches used to promote hiPSC-CMs maturation. In the second part, we focus on the evolving 3D culture model - it's structure, the effect on hiPSC-CMs maturation, incorporation with different maturation methods, limitations and future prospects.
  • Nawin Chanthra, Tomoyuki Abe, Matthew Miyamoto, Kiyotoshi Sekiguchi, Chulan Kwon, Yutaka Hanazono, Hideki Uosaki
    Scientific reports 10(1) 4249-4249 2020年3月6日  査読有り最終著者責任著者
    Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) hold great promise for disease modeling and drug discovery. However, PSC-CMs exhibit immature phenotypes in culture, and the lack of maturity limits their broad applications. While physical and functional analyses are generally used to determine the status of cardiomyocyte maturation, they could be time-consuming and often present challenges in comparing maturation-enhancing strategies. Therefore, there is a demand for a method to assess cardiomyocyte maturation rapidly and reproducibly. In this study, we found that Myomesin-2 (Myom2), encoding M-protein, is upregulated postnatally, and based on this, we targeted TagRFP to the Myom2 locus in mouse embryonic stem cells. Myom2-RFP+ PSC-CMs exhibited more mature phenotypes than RFP- cells in morphology, function and transcriptionally, conductive to sarcomere shortening assays. Using this system, we screened extracellular matrices (ECMs) and identified laminin-511/521 as potent enhancers of cardiomyocyte maturation. Together, we developed and characterized a novel fluorescent reporter system for the assessment of cardiomyocyte maturation and identified potent maturation-enhancing ECMs through this simple and rapid assay. This system is expected to facilitate use of PSC-CMs in a variety of scientific and medical investigations.
  • Mohamed Darwish, Hirofumi Nishizono, Hideki Uosaki, Hitomi Sawada, Taketaro Sadahiro, Masaki Ieda, Keizo Takao
    Journal of Neuroscience Methods 317 149-156 2019年4月  査読有り
  • Taketaro Sadahiro, Mari Isomi, Naoto Muraoka, Hidenori Kojima, Sho Haginiwa, Shota Kurotsu, Fumiya Tamura, Hidenori Tani, Shugo Tohyama, Jun Fujita, Hiroyuki Miyoshi, Yoshifumi Kawamura, Naoki Goshima, Yuka W Iwasaki, Kensaku Murano, Kuniaki Saito, Mayumi Oda, Peter Andersen, Chulan Kwon, Hideki Uosaki, Hirofumi Nishizono, Keiichi Fukuda, Masaki Ieda
    Cell stem cell 23(3) 382-395 2018年9月6日  査読有り
    The mesoderm arises from pluripotent epiblasts and differentiates into multiple lineages; however, the underlying molecular mechanisms are unclear. Tbx6 is enriched in the paraxial mesoderm and is implicated in somite formation, but its function in other mesoderms remains elusive. Here, using direct reprogramming-based screening, single-cell RNA-seq in mouse embryos, and directed cardiac differentiation in pluripotent stem cells (PSCs), we demonstrated that Tbx6 induces nascent mesoderm from PSCs and determines cardiovascular and somite lineage specification via its temporal expression. Tbx6 knockout in mouse PSCs using CRISPR/Cas9 technology inhibited mesoderm and cardiovascular differentiation, whereas transient Tbx6 expression induced mesoderm and cardiovascular specification from mouse and human PSCs via direct upregulation of Mesp1, repression of Sox2, and activation of BMP/Nodal/Wnt signaling. Notably, prolonged Tbx6 expression suppressed cardiac differentiation and induced somite lineages, including skeletal muscle and chondrocytes. Thus, Tbx6 is critical for mesoderm induction and subsequent lineage diversification.
  • Hiromasa Hara, Hiroaki Shibata, Kazuaki Nakano, Tomoyuki Abe, Hideki Uosaki, Takahiro Ohnuki, Shuji Hishikawa, Satoshi Kunita, Masahito Watanabe, Osamu Nureki, Hiroshi Nagashima, Yutaka Hanazono
    Experimental Animals 67(2) 139-146 2018年  査読有り
  • Gun-Sik Cho, Dong I. Lee, Emmanouil Tampakakis, Sean Murphy, Peter Andersen, Hideki Uosaki, Stephen Chelko, Khalid Chakir, Ingie Hong, Kinya Seo, Huei-Sheng Vincent Chen, Xiongwen Chen, Cristina Basso, Steven R. Houser, Gordon F. Tomaselli, Brian O'Rourke, Daniel P. Judge, David A. Kass, Chulan Kwon
    CELL REPORTS 18(2) 571-582 2017年1月  査読有り
  • Hideki Uosaki, Y-h Taguchi
    GENOMICS PROTEOMICS & BIOINFORMATICS 14(4) 207-215 2016年8月  査読有り筆頭著者責任著者
  • Hideki Uosaki, Patrick Cahan, Dong I. Lee, Songnan Wang, Matthew Miyamoto, Laviel Fernandez, David A. Kass, Chulan Kwon
    CELL REPORTS 13(8) 1705-1716 2015年11月  査読有り筆頭著者
  • Lincoln T. Shenje, Peter Andersen, Hideki Uosaki, Laviel Fernandez, Peter P. Rainer, Gun-Sik Cho, Dong-ik Lee, Weimin Zhong, Richard P. Harvey, David A. Kass, Chulan Kwon
    ELIFE 3 e02164 2014年4月  査読有り
  • Hideki Uosaki, Ajit Magadum, Kinya Seo, Hiroyuki Fukushima, Ayako Takeuchi, Yasuaki Nakagawa, Kara White Moyes, Genta Narazaki, Koichiro Kuwahara, Michael Laflamme, Satoshi Matsuoka, Norio Nakatsuji, Kazuwa Nakao, Chulan Kwon, David A. Kass, Felix B. Engel, Jun K. Yamashita
    Circulation: Cardiovascular Genetics 6(6) 624-633 2013年12月  査読有り筆頭著者
  • Kohei Yamamizu, Taichi Matsunaga, Hideki Uosaki, Hiroyuki Fukushima, Shiori Katayama, Mina Hiraoka-Kanie, Kohnosuke Mitani, Jun K. Yamashita
    Journal of Cell Biology 202(1) 179 2013年  査読有り
  • Hideki Uosaki, Peter Andersen, Lincoln T. Shenje, Laviel Fernandez, Sofie Lindgren Christiansen, Chulan Kwon
    PLOS ONE 7(10) e46413 2012年10月  査読有り筆頭著者
  • Hidetoshi Masumoto, Takehiko Matsuo, Kohei Yamamizu, Hideki Uosaki, Genta Narazaki, Shiori Katayama, Akira Marui, Tatsuya Shimizu, Tadashi Ikeda, Teruo Okano, Ryuzo Sakata, Jun K. Yamashita
    STEM CELLS 30(6) 1196-1205 2012年6月  査読有り
  • Peter Andersen, Hideki Uosaki, Lincoln T. Shenje, Chulan Kwon
    TRENDS IN CELL BIOLOGY 22(5) 257-265 2012年5月  査読有り筆頭著者
  • Hideki Uosaki, Hiroyuki Fukushima, Ayako Takeuchi, Satoshi Matsuoka, Norio Nakatsuji, Shinya Yamanaka, Jun K. Yamashita
    PLOS ONE 6(8) e23657 2011年8月  査読有り筆頭著者
  • Masataka Fujiwara, Peishi Yan, Tomomi G. Otsuji, Genta Narazaki, Hideki Uosaki, Hiroyuki Fukushima, Koichiro Kuwahara, Masaki Harada, Hiroyuki Matsuda, Satoshi Matsuoka, Keisuke Okita, Kazutoshi Takahashi, Masato Nakagawa, Tadashi Ikeda, Ryuzo Sakata, Christine L. Mummery, Norio Nakatsuji, Shinya Yamanaka, Kazuwa Nakao, Jun K. Yamashita
    PLOS ONE 6(2) e16734 2011年2月  査読有り
  • Kohei Yamamizu, Taichi Matsunaga, Hideki Uosaki, Hiroyuki Fukushima, Shiori Katayama, Mina Hiraoka-Kanie, Kohnosuke Mitani, Jun K. Yamashita
    JOURNAL OF CELL BIOLOGY 189(2) 325-338 2010年4月  査読有り
  • Shinobu Kuratomi, Yoko Ohmori, Masayuki Ito, Kuniko Shimazaki, Shin-ichi Muramatsu, Hiroaki Mizukami, Hideki Uosaki, Jun K. Yamashita, Yuji Arai, Koichiro Kuwahara, Makoto Takano
    CARDIOVASCULAR RESEARCH 83(4) 682-687 2009年9月  査読有り
  • Peishi Yan, Atsushi Nagasawa, Hideki Uosaki, Akihiro Sugimoto, Kohei Yamamizu, Mizue Teranishi, Hiroyuki Matsuda, Satoshi Matsuoka, Tadashi Ikeda, Masashi Komeda, Ryuzo Sakata, Jun K. Yamashita
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 379(1) 115-120 2009年1月  査読有り
  • Genta Narazaki, Hideki Uosaki, Mizue Teranishi, Keisuke Okita, Bongju Kim, Satoshi Matsuoka, Shinya Yamanaka, Jun K. Yamashita
    CIRCULATION 118(5) 498-506 2008年7月  査読有り
  • Kentoku Yanagi, Makoto Takano, Genta Narazaki, Hideki Uosaki, Takuhiro Hoshino, Takahiro Ishii, Takurou Misaki, Jun K. Yamashita
    STEM CELLS 25(11) 2712-2719 2007年11月  査読有り

MISC

 3

書籍等出版物

 5

講演・口頭発表等

 94

担当経験のある科目(授業)

 6

共同研究・競争的資金等の研究課題

 24