Updated on 2024/07/14

写真a

 
MATSUNAGA, Hiroko
 
Affiliation
Research Council (Research Organization), Research Organization for Nano & Life Innovation
Job title
Junior Researcher(Assistant Professor)
Degree
博士(理学) ( 大阪府立大学 )

Research Experience

  • 2018.07
    -
    Now

    Waseda University   Research Organization for Nano & Life Innovation

  • 2016.06
    -
    2018.06

    Waseda University

  • 1993.04
    -
    2018.06

    株式会社 日立製作所   中央研究所   研究員

  • 2001.02
    -
    2003.02

    アイオワ州立大学   客員研究員

  • 1994.04
    -
    1995.03

    Osaka University

Education Background

  • 1989.04
    -
    1993.03

    Osaka Prefecture University  

Research Areas

  • Molecular biology
 

Papers

  • 【あなたのラボから薬を生み出す アカデミア創薬の実践 All JAPAN体制の先端技術支援を利用した創薬の最前線】(第1章)最新の疾患標的分子の探索・評価技術 シングルセル/微小組織マルチオミクス解析

    由良 敬, 松永 浩子, 細川 正人, 和泉 自泰, 村松 知成, 福永 津嵩, 浜田 道昭, 馬場 健史, 竹山 春子

    実験医学   42 ( 2 ) 199 - 204  2024.02

     View Summary

    1つの細胞や微小組織からゲノムDNA,RNA,タンパク質,低分子代謝物などのオミクス情報を測定し,それらの情報を有機的に組み上げることで,細胞や組織の状態を理解する試みが急速に進められている.ここには最先端のバイオテクノロジーと情報解析方法が詰め込まれている.自ら明らかにしたいことが,どの手法を用いることで実現するかを知るためには,それぞれの解析手法がどのような特性をもっているのかを知らなければならない.その助けになることをめざして,ここではわれわれが展開するシングルセル/微小組織のオミクス解析を紹介する.(著者抄録)

  • Abstract P3026: Multimodal Imaging To Identify Pre-signals For Acute Aortic Dissection By Using Marfan Syndrome Model Mouse

    KAORI SUGIYAMA, Masahiro Ando, Hiroko Matsunaga, Ryota Wagatsuma, Haruki Uchino, Kenichi Kimura, Katja Schenke-layland, Makoto Arita, Hiromi Yanagisawa, Haruko Takeyama

    Circulation Research   133 ( Suppl_1 )  2023.08

     View Summary

    Background: Marfan syndrome (MFS) is a genetic disorder caused by mutations in the gene encoding theextracellular matrix Fibrillin-1 (FBN1), resulting in systemic connective tissue abnormalities. The mostcommon life-threatening conditions are aortic rupture and/or dissection. Aortic dissections show a highmortality rate, but the details of molecular triggers are unknown, particularly in acute aortic dissection(AAD). Raman spectroscopy is a technique for analyzing molecular structures from Raman spectra. It hasrecently attracted attention in the biology field because it can non-invasively distinguish between diseaseand non-disease areas.

    Objectives: This study aimed to identify the signals that induce changes in the aortic wall prior to acuteaortic dissection using genetic analysis and Raman spectroscopy with preservation of positional information.

    Methods: An acute aortic dissection model was created by using MFS model mice (Fbn1 mgR/mgR ) byadministration of angiotensin II (AngII) via osmotic pump (Fbn1 mgR/mgR -AAD). Aortas from wild-type(WT), Fbn1mgR/mgR and Fbn1mgR/mgR -AAD mice were harvested, and frozen sections were prepared. Thevascular wall of each mouse was assessed histologically, followed by spatial transcriptomic analysis andRaman microscopy measurements. Multivariate data analysis was performed on the obtained Ramanspectra. Mass spectrometry (MS) imaging was utilized for lipidome analysis.

    Results: Label-free imaging of extracellular substrates (collagen fibres, elastic fibres, versican, andaggrecan), lipids, and cells was produced in WT, Fbn1mgR/mgR and Fbn1mgR/mgR- AAD aortic tissue. Spatialtranscriptomic analysis identified differentially expressed genes among these aortas. We identified markedlipid accumulations in the acute aortic dissection models with Raman and MS imaging.

    DOI

  • Linking antigen specific T-cell dynamics in a microfluidic chip to single cell transcription patterns.

    Hiroki Ide, Taiki Aoshi, Masato Saito, Wilfred Villariza Espulgar, Jonathan Campos Briones, Masahito Hosokawa, Hiroko Matsunaga, Koji Arikawa, Haruko Takeyama, Shohei Koyama, Hyota Takamatsu, Eiichi Tamiya

    Biochemical and biophysical research communications   657   8 - 15  2023.03  [International journal]

     View Summary

    A new non-invasive screening profile has been realized that can aid in determining T-cell activation state at single-cell level. Production of activated T-cells with good specificity and stable proliferation is greatly beneficial for advancing adoptive immunotherapy as innate immunological cells are not effective in recognizing and eliminating cancer as expected. The screening method is realized by relating intracellular Ca2+ intensity and motility of T-cells interacting with APC (Antigen Presenting Cells) in a microfluidic chip. The system is tested using APC pulsed with OVA257-264 peptide and its modified affinities (N4, Q4, T4 and V4), and the T-cells from OT-1 mice. In addition, single cell RNA sequencing reveals the activation states of the cells and the clusters from the derived profiles can be indicative of the T-cell activation state. The presented system here can be versatile for a comprehensive application to proceed with T-cell-based immunotherapy and screen the antigen-specific T-cells with excellent efficiency and high proliferation.

    DOI PubMed

    Scopus

    3
    Citation
    (Scopus)
  • Application of organoid culture from HPV18-positive small cell carcinoma of the uterine cervix for precision medicine.

    Misako Kusakabe, Ayumi Taguchi, Michihiro Tanikawa, Daisuke Hoshi, Saki Tsuchimochi, Xi Qian, Yusuke Toyohara, Akira Kawata, Ryota Wagatsuma, Kohei Yamaguchi, Yoko Yamamoto, Masako Ikemura, Kenbun Sone, Mayuyo Mori-Uchino, Hiroko Matsunaga, Tetsushi Tsuruga, Takeshi Nagamatsu, Iwao Kukimoto, Osamu Wada-Hiraike, Masahito Kawazu, Tetsuo Ushiku, Haruko Takeyama, Katsutoshi Oda, Kei Kawana, Yoshitaka Hippo, Yutaka Osuga

    Cancer medicine   12 ( 7 ) 8476 - 8489  2023.01  [International journal]

     View Summary

    BACKGROUND: Small cell carcinoma of the uterine cervix (SCCC) is a rare and highly malignant human papillomavirus (HPV)-associated cancer in which human genes related to the integration site can serve as a target for precision medicine. The aim of our study was to establish a workflow for precision medicine of HPV-associated cancer using patient-derived organoid. METHODS: Organoid was established from the biopsy of a patient diagnosed with HPV18-positive SCCC. Therapeutic targets were identified by whole exome sequencing (WES) and RNA-seq analysis. Drug sensitivity testing was performed using organoids and organoid-derived mouse xenograft model. RESULTS: WES revealed that both the original tumor and organoid had 19 somatic variants in common, including the KRAS p.G12D pathogenic variant. Meanwhile, RNA-seq revealed that HPV18 was integrated into chromosome 8 at 8q24.21 with increased expression of the proto-oncogene MYC. Drug sensitivity testing revealed that a KRAS pathway inhibitor exerted strong anti-cancer effects on the SCCC organoid compared to a MYC inhibitor, which were also confirmed in the xenograft model. CONCLUSION: In this study, we confirmed two strategies for identifying therapeutic targets of HPV-derived SCCC, WES for identifying pathogenic variants and RNA sequencing for identifying HPV integration sites. Organoid culture is an effective tool for unveiling the oncogenic process of rare tumors and can be a breakthrough for the development of precision medicine for patients with HPV-positive SCCC.

    DOI PubMed

    Scopus

    5
    Citation
    (Scopus)
  • Protective Role of Endothelial Fibulin‐4 in Valvulo‐Arterial Integrity

    Tram Anh Vu Nguyen, Caroline Antunes Lino, Huynh Thuy Hang, Juliano Vilela Alves, Bui Quoc Thang, Seung Jae Shin, Kaori Sugiyama, Hiroko Matsunaga, Haruko Takeyama, Yoshito Yamashiro, Hiromi Yanagisawa

    Journal of the American Heart Association   12 ( 1 )  2023.01

     View Summary

    Background

    <p lang="en"> Homeostasis of the vessel wall is cooperatively maintained by endothelial cells (ECs), smooth muscle cells, and adventitial fibroblasts. The genetic deletion of fibulin‐4 ( Fbln4 ) in smooth muscle cells ( SMKO ) leads to the formation of thoracic aortic aneurysms with the disruption of elastic fibers. Although Fbln4 is expressed in the entire vessel wall, its function in ECs and relevance to the maintenance of valvulo‐arterial integrity are not fully understood.

    </p> Methods and Results

    <p lang="en"> Gene silencing of FBLN4 was conducted on human aortic ECs to evaluate morphological changes and gene expression profile. Fbln4 double knockout ( DKO ) mice in ECs and smooth muscle cells were generated and subjected to histological analysis, echocardiography, Western blotting, RNA sequencing, and immunostaining. An evaluation of the thoracic aortic aneurysm phenotype and screening of altered signaling pathways were performed. Knockdown of FBLN4 in human aortic ECs induced mesenchymal cell–like changes with the upregulation of mesenchymal genes, including TAGLN and MYL9 . DKO mice showed the exacerbation of thoracic aortic aneurysms when compared with those of SMKO and upregulated Thbs1, a mechanical stress–responsive molecule, throughout the aorta. DKO mice also showed progressive aortic valve thickening with collagen deposition from postnatal day 14, as well as turbulent flow in the ascending aorta. Furthermore, RNA sequencing and immunostaining of the aortic valve revealed the upregulation of genes involved in endothelial‐to‐mesenchymal transition, inflammatory response, and tissue fibrosis in DKO valves and the presence of activated valve interstitial cells.

    </p> Conclusions

    <p lang="en">The current study uncovers the pivotal role of endothelial fibulin‐4 in the maintenance of valvulo‐arterial integrity, which influences thoracic aortic aneurysm progression.

    </p>

    DOI

  • Cells with stem‐like properties are associated with the development of <scp>HPV18</scp> ‐positive cervical cancer

    Misako Kusakabe, Ayumi Taguchi, Michihiro Tanikawa, Ryota Wagatsuma, Miki Yamazaki, Saki Tsuchimochi, Yusuke Toyohara, Akira Kawata, Satoshi Baba, Toshihide Ueno, Kenbun Sone, Mayuyo Mori‐Uchino, Masako Ikemura, Hiroko Matsunaga, Takeshi Nagamatsu, Osamu Wada‐Hiraike, Masahito Kawazu, Tetsuo Ushiku, Haruko Takeyama, Katsutoshi Oda, Kei Kawana, Hiroyuki Mano, Yutaka Osuga

    Cancer Science   114 ( 3 ) 885 - 895  2022.12

    DOI

    Scopus

    4
    Citation
    (Scopus)
  • Integrated spatial analysis of gene mutation and gene expression for understanding tumor diversity in formalin-fixed paraffin-embedded lung adenocarcinoma

    Miki Yamazaki, Masahito Hosokawa, Hiroko Matsunaga, Koji Arikawa, Kazuya Takamochi, Kenji Suzuki, Takuo Hayashi, Hideki Kambara, Haruko Takeyama

    Frontiers in Oncology   12  2022.11

     View Summary

    Introduction

    A deeper understanding of intratumoral heterogeneity is essential for prognosis prediction or accurate treatment plan decisions in clinical practice. However, due to the cross-links and degradation of biomolecules within formalin-fixed paraffin-embedded (FFPE) specimens, it is challenging to analyze them. In this study, we aimed to optimize the simultaneous extraction of mRNA and DNA from microdissected FFPE tissues (φ = 100 µm) and apply the method to analyze tumor diversity in lung adenocarcinoma before and after erlotinib administration.

    Method

    Two magnetic beads were used for the simultaneous extraction of mRNA and DNA. The decross-linking conditions were evaluated for gene mutation and gene expression analyses of microdissected FFPE tissues. Lung lymph nodes before treatment and lung adenocarcinoma after erlotinib administration were collected from the same patient and were preserved as FFPE specimens for 4 years. Gene expression and gene mutations between histologically classified regions of lung adenocarcinoma (pre-treatment tumor in lung lymph node biopsies and post-treatment tumor, normal lung, tumor stroma, and remission stroma, in resected lung tissue) were compared in a microdissection-based approach.

    Results

    Using the optimized simultaneous extraction of DNA and mRNA and whole-genome amplification, we detected approximately 4,000–10,000 expressed genes and the epidermal growth factor receptor (EGFR) driver gene mutations from microdissected FFPE tissues. We found the differences in the highly expressed cancer-associated genes and the positive rate of EGFR exon 19 deletions among the tumor before and after treatment and tumor stroma, even though they were collected from tumors of the same patient or close regions of the same specimen.

    Conclusion

    Our integrated spatial analysis method would be applied to various FFPE pathology specimens providing area-specific gene expression and gene mutation information.

    DOI

    Scopus

    2
    Citation
    (Scopus)
  • Reproducible and sensitive micro-tissue RNA-sequencing from formalin-fixed paraffin-embedded tissue for spatial gene expression analysis

    Hiroko Matsunaga, Koji Arikawa, Miki Yamazaki, Ryota Wagatsuma, Keigo Ide, Samuel Ashok Zachariah, Kazuya Takamochi, Kenji Suzuki, Takuo Hayashi, Masahito Hosokawa, Hideki Kambara, Haruko Takeyama

    Scientific Reports   12 ( 1 )  2022.03

     View Summary

    Abstract

    Spatial transcriptome analysis of formalin-fixed paraffin-embedded (FFPE) tissues using RNA-sequencing (RNA-seq) provides interactive information on morphology and gene expression, which is useful for clinical applications. However, despite the advantages of long-term storage at room temperature, FFPE tissues may be severely damaged by methylene crosslinking and provide less gene information than fresh-frozen tissues. In this study, we proposed a sensitive FFPE micro-tissue RNA-seq method that combines the punching of tissue sections (diameter: 100 μm) and the direct construction of RNA-seq libraries. We evaluated a method using mouse liver tissues at 2 years after fixation and embedding and detected approximately 7,000 genes in micro-punched tissue-spots (thickness: 10 μm), similar to that detected with purified total RNA (2.5 ng) equivalent to the several dozen cells in the spot. We applied this method to clinical FFPE specimens of lung cancer that had been fixed and embedded 6 years prior, and found that it was possible to determine characteristic gene expression in the microenvironment containing tumor and non-tumor cells of different morphologies. This result indicates that spatial gene expression analysis of the tumor microenvironment is feasible using FFPE tissue sections stored for extensive periods in medical facilities.

    DOI

  • Identification of two cancer stem cell-like populations in triple-negative breast cancer xenografts

    Jun Nakayama, Hiroko Matsunaga, Koji Arikawa, Takuya Yoda, Masahito Hosokawa, Haruko Takeyama, Yusuke Yamamoto, Kentaro Semba

       2021.10

     View Summary

    Abstract

    Gene expression analysis at the single-cell level by next generation sequencing has revealed the existence of clonal dissemination and microheterogeneity in cancer metastasis. The current spatial analysis technologies can elucidate the heterogeneity of cell–cell interactions in situ. To reveal the regional and expressional heterogeneity in primary tumors and metastases, we performed transcriptomic analysis of microtissues dissected from a triple-negative breast cancer (TNBC) cell line MDA-MB-231 xenograft model with our automated tissue microdissection punching technology. This multiple-microtissue transcriptome analysis revealed three cancer cell-type clusters in the primary tumor and axillary lymph node metastasis, two of which were cancer stem cell (CSC)-like clusters (CD44/MYC-high, HMGA1-high). Reanalysis of public single-cell RNA-seq (scRNA-seq) datasets confirmed that the two CSC-like populations existed both in TNBC xenograft models and TNBC patients. In addition, the gene signature of the HMGA1-high CSC-like cluster has the potential to serve as a novel biomarker for diagnosis. The diversity of these multiple CSC-like populations may cause differential anticancer drug resistance, increasing the difficulty of curing this cancer.

    DOI

  • Distinctive Regulation of Emotional Behaviors and Fear-Related Gene Expression Responses in Two Extended Amygdala Subnuclei With Similar Molecular Profiles

    Shuhei Ueda, Masahito Hosokawa, Koji Arikawa, Kiyofumi Takahashi, Mao Fujiwara, Manami Kakita, Taro Fukada, Hiroaki Koyama, Shin-ichiro Horigane, Keiichi Itoi, Masaki Kakeyama, Hiroko Matsunaga, Haruko Takeyama, Haruhiko Bito, Sayaka Takemoto-Kimura

    Frontiers in Molecular Neuroscience   14  2021.09

     View Summary

    The central nucleus of the amygdala (CeA) and the lateral division of the bed nucleus of the stria terminalis (BNST) are the two major nuclei of the central extended amygdala that plays essential roles in threat processing, responsible for emotional states such as fear and anxiety. While some studies suggested functional differences between these nuclei, others showed anatomical and neurochemical similarities. Despite their complex subnuclear organization, subnuclei-specific functional impact on behavior and their underlying molecular profiles remain obscure. We here constitutively inhibited neurotransmission of protein kinase C-δ-positive (PKCδ+) neurons—a major cell type of the lateral subdivision of the CeA (CeL) and the oval nucleus of the BNST (BNSTov)—and found striking subnuclei-specific effects on fear- and anxiety-related behaviors, respectively. To obtain molecular clues for this dissociation, we conducted RNA sequencing in subnuclei-targeted micropunch samples. The CeL and the BNSTov displayed similar gene expression profiles at the basal level; however, both displayed differential gene expression when animals were exposed to fear-related stimuli, with a more robust expression change in the CeL. These findings provide novel insights into the molecular makeup and differential engagement of distinct subnuclei of the extended amygdala, critical for regulation of threat processing.

    DOI

    Scopus

    5
    Citation
    (Scopus)
  • Cortical transcriptome analysis after spinal cord injury reveals the regenerative mechanism of central nervous system in CRMP2 knock-in mice.

    Ayaka Sugeno, Wenhui Piao, Miki Yamazaki, Kiyofumi Takahashi, Koji Arikawa, Hiroko Matsunaga, Masahito Hosokawa, Daisuke Tominaga, Yoshio Goshima, Haruko Takeyama, Toshio Ohshima

    Neural regeneration research   16 ( 7 ) 1258 - 1265  2021.07  [International journal]

     View Summary

    Recent studies have shown that mutation at Ser522 causes inhibition of collapsin response mediator protein 2 (CRMP2) phosphorylation and induces axon elongation and partial recovery of the lost sensorimotor function after spinal cord injury (SCI). We aimed to reveal the intracellular mechanism in axotomized neurons in the CRMP2 knock-in (CRMP2KI) mouse model by performing transcriptome analysis in mouse sensorimotor cortex using micro-dissection punching system. Prior to that, we analyzed the structural pathophysiology in axotomized or neighboring neurons after SCI and found that somatic atrophy and dendritic spine reduction in sensorimotor cortex were suppressed in CRMP2KI mice. Further analysis of the transcriptome has aided in the identification of four hemoglobin genes Hba-a1, Hba-a2, Hbb-bs, and Hbb-bt that are significantly upregulated in wild-type mice with concomitant upregulation of genes involved in the oxidative phosphorylation and ribosomal pathways after SCI. However, we observed substantial upregulation in channel activity genes and downregulation of genes regulating vesicles, synaptic function, glial cell differentiation in CRMP2KI mice. Moreover, the transcriptome profile of CRMP2KI mice has been discussed wherein energy metabolism and neuronal pathways were found to be differentially regulated. Our results showed that CRMP2KI mice displayed improved SCI pathophysiology not only via microtubule stabilization in neurons, but also possibly via the whole metabolic system in the central nervous system, response changes in glial cells, and synapses. Taken together, we reveal new insights on SCI pathophysiology and the regenerative mechanism of central nervous system by the inhibition of CRMP2 phosphorylation at Ser522. All these experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee at Waseda University, Japan (2017-A027 approved on March 21, 2017; 2018-A003 approved on March 25, 2018; 2019-A026 approved on March 25, 2019).

    DOI PubMed

    Scopus

    7
    Citation
    (Scopus)
  • Development of an Inflammatory CD14+ Dendritic Cell Subset in Humanized Mice.

    Ryutaro Iwabuchi, Keigo Ide, Kazutaka Terahara, Ryota Wagatsuma, Rieko Iwaki, Hiroko Matsunaga, Yasuko Tsunetsugu-Yokota, Haruko Takeyama, Yoshimasa Takahashi

    Frontiers in immunology   12   643040 - 643040  2021  [International journal]

     View Summary

    Humanized mouse models are attractive experimental models for analyzing the development and functions of human dendritic cells (DCs) in vivo. Although various types of DC subsets, including DC type 3 (DC3s), have been identified in humans, it remains unclear whether humanized mice can reproduce heterogeneous DC subsets. CD14, classically known as a monocyte/macrophage marker, is reported as an indicator of DC3s. We previously observed that some CD14+ myeloid cells expressed CD1c, a pan marker for bona fide conventional DC2 (cDC2s), in humanized mouse models in which human FLT3L and GM-CSF genes were transiently expressed using in vivo transfection (IVT). Here, we aimed to elucidate the identity of CD14+CD1c+ DC-like cells in humanized mouse models. We found that CD14+CD1c+ cells were phenotypically different from cDC2s; CD14+CD1c+ cells expressed CD163 but not CD5, whereas cDC2s expressed CD5 but not CD163. Furthermore, CD14+CD1c+ cells primed and polarized naïve CD4+ T cells toward IFN-γ+ Th1 cells more profoundly than cDC2s. Transcriptional analysis revealed that CD14+CD1c+ cells expressed several DC3-specific transcripts, such as CD163, S100A8, and S100A9, and were clearly segregated from cDC2s and monocytes. When lipopolysaccharide was administered to the humanized mice, the frequency of CD14+CD1c+ cells producing IL-6 and TNF-α was elevated, indicating a pro-inflammatory signature. Thus, humanized mice are able to sustain development of functional CD14+CD1c+ DCs, which are equivalent to DC3 subset observed in humans, and they could be useful for analyzing the development and function of DC3s in vivo.

    DOI PubMed

    Scopus

    4
    Citation
    (Scopus)
  • Effective microtissue RNA extraction coupled with Smart-seq2 for reproducible and robust spatial transcriptome analysis.

    Miki Yamazaki, Masahito Hosokawa, Koji Arikawa, Kiyofumi Takahashi, Chikako Sakanashi, Takuya Yoda, Hiroko Matsunaga, Haruko Takeyama

    Scientific reports   10 ( 1 ) 7083 - 7083  2020.04  [Refereed]  [International journal]

     View Summary

    Spatial transcriptomics is useful for understanding the molecular organization of a tissue and providing insights into cellular function in a morphological context. In order to obtain reproducible results in spatial transcriptomics, we have to maintain tissue morphology and RNA molecule stability during the image acquisition and biomolecule collection processes. Here, we developed a tissue processing method for robust and reproducible RNA-seq from tissue microdissection samples. In this method, we suppressed RNA degradation in fresh-frozen tissue specimens by dehydration fixation and effectively collected a small amount of RNA molecules from microdissection samples by magnetic beads. We demonstrated the spatial transcriptome analysis of the mouse liver and brain in serial microdissection samples (100 μm in a diameter and 10 μm in thickness) produced by a microdissection punching system. Using our method, we could prevent RNA degradation at room temperature and effectively produce a sequencing library with Smart-seq2. This resulted in reproducible sequence read mapping in exon regions and the detection of more than 2000 genes compared to non-fixed samples in the RNA-seq analysis. Our method would be applied to various transcriptome analyses, providing the information for region specific gene expression in tissue specimens.

    DOI PubMed

    Scopus

    15
    Citation
    (Scopus)
  • Highly sensitive mutation quantification by high-dynamic-range capillary-array electrophoresis (HiDy CE).

    Takashi Anazawa, Hiroko Matsunaga, Shuhei Yamamoto, Ryoji Inaba

    Lab on a chip   20 ( 6 ) 1083 - 1091  2020.03  [Refereed]  [International journal]

     View Summary

    A simple and robust ultra-small four-color-fluorescence detection system was developed by integrating its components, namely, a four-capillary array, an injection-molded-plastic four-lens array, a four-dichroic-mirror array, and a CMOS sensor, as one device. The developed system was applied to a high-dynamic-range capillary-array electrophoresis (HiDy CE) to quantify a rare EGFR mutant (MT) of exon 19 deletion in a large excess of EGFR wild type (WT). Samples with serially diluted MT and constant-concentration WT were co-amplified by competitive PCR and subjected to HiDy CE. The MT peak in each electropherogram was then compared to the WT peak. As a result, MT was quantified with high-sensitivity (LOD of 0.004% MT/WT) and four-orders-of-magnitude dynamic range (0.01-100% MT/WT) by HiDy CE. Moreover, compared with existing methods, HiDy CE achieves higher speed, higher sample throughput, and lower consumable cost per sample. It has therefore great potential to be used in clinical practice.

    DOI PubMed

    Scopus

    5
    Citation
    (Scopus)
  • Combinatory use of distinct single-cell RNA-seq analytical platforms reveals the heterogeneous transcriptome response.

    Yukie Kashima, Ayako Suzuki, Ying Liu, Masahito Hosokawa, Hiroko Matsunaga, Masataka Shirai, Kohji Arikawa, Sumio Sugano, Takashi Kohno, Haruko Takeyama, Katsuya Tsuchihara, Yutaka Suzuki

    Scientific reports   8 ( 1 ) 3482 - 3482  2018.02  [Refereed]  [International journal]

     View Summary

    Single-cell RNA-seq is a powerful tool for revealing heterogeneity in cancer cells. However, each of the current single-cell RNA-seq platforms has inherent advantages and disadvantages. Here, we show that combining the different single-cell RNA-seq platforms can be an effective approach to obtaining complete information about expression differences and a sufficient cellular population to understand transcriptional heterogeneity in cancers. We demonstrate that it is possible to estimate missing expression information. We further demonstrate that even in the cases where precise information for an individual gene cannot be inferred, the activity of given transcriptional modules can be analyzed. Interestingly, we found that two distinct transcriptional modules, one associated with the Aurora kinase gene and the other with the DUSP gene, are aberrantly regulated in a minor population of cells and may thus contribute to the possible emergence of dormancy or eventual drug resistance within the population.

    DOI PubMed

    Scopus

    15
    Citation
    (Scopus)
  • 腫瘍内微小不均一性解明のための空間トランスクリプトミクス解析技術の確立

    中山 淳, 有川 浩司, 丸山 徹, 松永 浩子, 依田 卓也, 細川 正人, 神原 秀記, 竹山 春子, 仙波 憲太郎

    生命科学系学会合同年次大会   2017年度   [4AT27 - 1362)]  2017.12

  • A highly sensitive and accurate gene expression analysis by sequencing ("bead-seq") for a single cell.

    Hiroko Matsunaga, Mari Goto, Koji Arikawa, Masataka Shirai, Hiroyuki Tsunoda, Huan Huang, Hideki Kambara

    Analytical biochemistry   471   9 - 16  2015.02  [Refereed]  [International journal]

     View Summary

    Analyses of gene expressions in single cells are important for understanding detailed biological phenomena. Here, a highly sensitive and accurate method by sequencing (called "bead-seq") to obtain a whole gene expression profile for a single cell is proposed. A key feature of the method is to use a complementary DNA (cDNA) library on magnetic beads, which enables adding washing steps to remove residual reagents in a sample preparation process. By adding the washing steps, the next steps can be carried out under the optimal conditions without losing cDNAs. Error sources were carefully evaluated to conclude that the first several steps were the key steps. It is demonstrated that bead-seq is superior to the conventional methods for single-cell gene expression analyses in terms of reproducibility, quantitative accuracy, and biases caused during sample preparation and sequencing processes.

    DOI PubMed

    Scopus

    15
    Citation
    (Scopus)
  • Non-biased and efficient global amplification of a single-cell cDNA library.

    Huan Huang, Mari Goto, Hiroyuki Tsunoda, Lizhou Sun, Kiyomi Taniguchi, Hiroko Matsunaga, Hideki Kambara

    Nucleic acids research   42 ( 2 ) e12  2014.01  [Refereed]  [International journal]

     View Summary

    Analysis of single-cell gene expression promises a more precise understanding of molecular mechanisms of a living system. Most techniques only allow studies of the expressions for limited numbers of gene species. When amplification of cDNA was carried out for analysing more genes, amplification biases were frequently reported. A non-biased and efficient global-amplification method, which uses a single-cell cDNA library immobilized on beads, was developed for analysing entire gene expressions for single cells. Every step in this analysis from reverse transcription to cDNA amplification was optimized. By removing degrading excess primers, the bias due to the digestion of cDNA was prevented. Since the residual reagents, which affect the efficiency of each subsequent reaction, could be removed by washing beads, the conditions for uniform and maximized amplification of cDNAs were achieved. The differences in the amplification rates for randomly selected eight genes were within 1.5-folds, which could be negligible for most of the applications of single-cell analysis. The global amplification gives a large amount of amplified cDNA (>100 μg) from a single cell (2-pg mRNA), and that amount is enough for downstream analysis. The proposed global-amplification method was used to analyse transcript ratios of multiple cDNA targets (from several copies to several thousand copies) quantitatively.

    DOI PubMed

    Scopus

    19
    Citation
    (Scopus)
  • Integrated on-capillary instrumentation for gene expression measurement directly from cells.

    Hiroko Matsunaga, Takashi Anazawa, Edward S Yeung

    Electrophoresis   24 ( 3 ) 458 - 65  2003.01  [Refereed]  [International journal]

     View Summary

    We studied the fundamental instrumental issues relevant to a capillary-based integrated system to measure expression of a specific gene directly from cells. Samples were introduced into a capillary by use of a syringe pump. All reactions were carried out in a microthermocycler, where a part of the capillary having 1 microL inner volume was used as a reaction vessel. First, cells were lysed by heating to release RNA, followed by deoxyribonuclease (DNase) treatment. Then, reverse transcription-polymerase chain reaction (RT-PCR) was performed to obtain amplified products from the targeted mRNA. Finally, the product was verified by capillary electrophoresis (CE) with laser-induced fluorescence detection. The whole protocol was completed in the system in 3 h. PCR product from beta-actin mRNA in 16 human lymphoblast cells was obtained with a signal-to-noise ratio (S/N) of 3400 +/- 730 (n = 3). Therefore, the system is reproducible and sensitive enough to measure gene expression from a single cell. We show that the amplified fragment from breast cancer-specific mRNA was obtained from cells of breast cancer cell line, but was not obtained from cells of hepatoma cell line. These results therefore lay the foundations for future CE or microchip instrumentation for high-throughput automated gene-expression analysis.

    DOI PubMed

    Scopus

    16
    Citation
    (Scopus)
  • Electrophoretic quantitation of nucleic acids without amplification by single-molecule imaging.

    Takashi Anazawa, Hiroko Matsunaga, Edward S Yeung

    Analytical chemistry   74 ( 19 ) 5033 - 8  2002.10  [Refereed]  [International journal]

     View Summary

    We have developed a novel high-performance quantitative assay for unamplified nucleic acids that is based on single-molecule imaging. The apparatus is a simple but highly sensitive single-molecule detection system that uses a normal CCD camera instead of an image-intensified CCD camera. After the DNA molecules in a sample were labeled with YOYO-1, they were induced to migrate electrophoretically in a polymer solution and imaged. No chemical or biochemical amplification was required. Direct quantitation of the sample by counting molecules was possible, because the number counted over the measurement period was directly proportional to the concentration of DNA molecules in the sample. Nonspecifically labeled impurities that would degrade the sensitivity of the assay were successfully reduced and discriminated from the DNA molecules by differences in electrophoretic mobility. By using beta-actin DNA (838 bp) as a model sample, we demonstrate that this protocol was fast (10-min measurement period), highly sensitive (limit of quantitation: approximately 10(3) copies/sample, or 3 x 10(-16) M), quantitative, and covered a wide linear dynamic range (approximately 10(4)). This high-performance assay promises to be a powerful technology for the quantitation of specific varieties of mRNA in the study of gene functions and diseases and in the clinical detection of mutant cells.

    DOI PubMed

    Scopus

    49
    Citation
    (Scopus)
  • Application of differential display to identify genes for lung cancer detection in peripheral blood.

    Hiroko Matsunaga, Nanae Hangai, Yoshimasa Aso, Kazunori Okano, Masafumi Kawamura, Kouichi Kobayashi, Hideki Kambara, Jeff H Hoger, Masato Mitsuhashi

    International journal of cancer   100 ( 5 ) 592 - 9  2002.08  [Refereed]  [International journal]

     View Summary

    A blood assay for detection of lung cancer biomarkers could significantly improve cancer patient prognosis and survival rates. Amplified fragment length polymorphism-differential display (AFLP-DD) was used to identify gene transcripts found in lung cancer tissue and the peripheral blood of lung cancer patients. The clones were evaluated for gene expression in lung cancer tissue, peripheral blood of lung cancer patients and healthy volunteers' blood. The isolated gene transcript clones were found to be from the syndecan 1 gene, collagen 1 gene and 2 novel genes. All 4 transcripts were expressed in normal lung tissue, 4 cultured primary lung cells and 6 lung cancer cell lines. RNA was isolated from peripheral blood samples of 69 lung cancer patients. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to test for the presence of cytokeratin 19 and the 4 gene mRNA transcripts in blood RNA. The positive detection rate of at least 1 of the 5 transcripts was 79% for lung adenocarcinoma and 62% for squamous carcinoma. Using RT-PCR, at least 1 of the markers was found in 53% of stage I patients, 100% of stage II, 71% of stage III and 81% of stage IV lung cancer patients. Blood samples from 20 healthy volunteers were also tested, but only 1 of the 5 transcripts was found in 1 patient. These new molecular markers may aid early detection, staging and follow-up of lung cancer patients by RNA isolated from blood.

    DOI PubMed

    Scopus

    17
    Citation
    (Scopus)
  • Characteristics of selective polymerase chain reaction (PCR) using two-base anchored primers and improvement of its specificity

    K Okano, C Uematsu, H Matsunaga, H Kambara

    ELECTROPHORESIS   19 ( 18 ) 3071 - 3078  1998.12  [Refereed]

     View Summary

    We have developed a reliable method for eliminating base-mispair amplification in selective polymerase chain reaction (PCR), which is utilized for amplifying unknown sequence fragments produced by restriction enzyme reaction. The proposed procedure applies amplified fragment length polymorphism (AFLP) with high fidelity. Selective PCR utilizes the known polymerase reaction characteristic that the complementary strand extension is strongly affected by matching a template with the 3'-terminus of the primers. However, false positive amplification is frequently observed because the specificity of terminal bases for discrimination of fragments (usually, 1-3 anchor sequences) is not enough to separate each fragment. A protocol for the selective PCR separation of every fragment was therefore investigated. A single-base mismatch was artificially introduced on the 4th base position from the 3' end of the primers to improve the hybridization specificity of anchored 2-bases at the 3' termini of primers. PCR reaction was carried out at 66 degrees C to prevent false positive amplification. The concentration of the primers having anchored-base sequences of AA, AT, TA, and TT must be three times larger than that of other primers because the T-m values for these sequences are lower than the others. As all the fragments can be separated into groups with high fidelity, the improved selective PCR will be applied to gene finding and analyzing differences on genome sequences based on AFLP.

    DOI PubMed

    Scopus

    9
    Citation
    (Scopus)
  • Selecting and amplifying one fragment from a DNA fragment mixture by polymerase chain reaction with a pair of selective primers

    H Matsunaga, Y Kohara, K Okano, H Kambara

    ELECTROPHORESIS   17 ( 12 ) 1833 - 1840  1996.12  [Refereed]

     View Summary

    A new method for selecting and amplifying a single DNA fragment from a mixture is proposed. This method is applicable for the rapid classification of DNA fragments from a mixture and for preparation of sequencing templates. DNAs of several to tens of kilobases (kb) are digested with a four-base recognition restriction enzyme to produce smaller fragments. The complementary strand extension reactions are then carried out to produce fluorophore-labeled DNA fragments from the digestion products. These fragments can be rapidly classified according to their terminal-base sequences and their sizes are analyzed by capillary-array gel electrophoresis (CAGE). Electropherograms are used to characterize the fragments and to select polymerase chain reaction (PCR) primers. Any fragment in a digestion mixture can be amplified by PCR with a pair of primers selected from a primer pool by referring to the electropherograms of the fragments. This method was successfully used to compare the electropherograms of two different DNA strands and to sequence a several-kb DNA fragment without subcloning. Combined with CAGE, this method could be used to dramatically simplify DNA fragment analysis.

    DOI PubMed

    Scopus

    2
    Citation
    (Scopus)

▼display all

Research Projects

  • 次世代トランスクリプトーム解析を用いた肺癌個別化医療開発

    日本学術振興会  科学研究費助成事業

    Project Year :

    2022.04
    -
    2026.03
     

    林 大久生, 高阪 真路, 松永 浩子, 高持 一矢, 村川 泰裕, 北野 滋久

Misc

  • 遺伝子マーカーを用いた血中浮遊肺癌細胞の検出法の確立とその臨床応用

    松永浩子, 岡野和宣, 半谷七重, 川村雅文, HOGER J H, 神原秀記, 三橋将人

    日本分子生物学会年会プログラム・講演要旨集   23rd  2000

    J-GLOBAL

  • 遺伝子マーカーによる肺がん微小転移スクリーニング

    松永浩子, CHEN J, 岡野和宣, 半谷七重, 桑原克之, 川村雅文, QIN S, 三橋将人

    日本分子生物学会年会プログラム・講演要旨集   22nd  1999

    J-GLOBAL

  • Gene Discovery of Micro Metastasis of Human Lung Cancer Cells

    MATSUNAGA Hiroko, OKANO Kazunori, MITSUHASHI Masato, KUWABARA Katsuyuki, KAWAMURA Masahumi, KAMBARA Hideki

      21   335 - 335  1998.12

    CiNii

  • Characteristics of selective PCR using two-base anchored primers and improvement of its specificity

    OKANO K., UEMATSU C., MATSUNAGA H., KAMBARA H.

      21   339 - 339  1998.12

    CiNii

  • Subcloning Free Sequencing Method for Several kb DNAs using Selective Primers.

    松永浩子, 岡野和宣, 神原秀記

    日本分子生物学会年会プログラム・講演要旨集   20th  1997

    J-GLOBAL

  • High resolution analysis for DNA fragments by classifying fragments according to their terminal sequences using selective primers.

    岡野和宣, 植松千宗, 松永浩子, 神原秀記

    日本分子生物学会年会プログラム・講演要旨集   20th  1997

    J-GLOBAL

  • フラグメントウォーキング法による大容量DNA解析技術の開発 (4) 50Kb DNAの迅速配列決定法

    岡野和宣, 松永浩子, 村川克二, 神原秀記

    日本分子生物学会年会プログラム・講演要旨集   19th  1996

    J-GLOBAL

  • フラグメントウォーキング法による大容量DNA解析技術の開発 (3) 選択プライマーによる伸長反応の温度依存性の検討

    松永浩子, 岡野和宣, 神原秀記

    日本分子生物学会年会プログラム・講演要旨集   19th  1996

    J-GLOBAL

▼display all

Industrial Property Rights

  • 細胞塊微細分画デバイスおよび解析方法

    松永 浩子, 田邉 麻衣子, 有川 浩司

    Patent

    J-GLOBAL

  • 環状型一本鎖核酸、およびその調製方法と使用方法

    松永 浩子, 田邉 麻衣子

    Patent

    J-GLOBAL

  • 核酸増幅反応後の反応液の解析方法、解析装置及び核酸増幅反応後の反応液処理装置

    松永 浩子, 梶山 智晴, 太田 真理, 神原 秀記

    Patent

    J-GLOBAL

  • 細胞採取システム

    白井 正敬, 角田 弘之, 松永 浩子, 内田 憲孝

    Patent

    J-GLOBAL

  • 細胞採取システム

    特許第5487152号

    白井 正敬, 角田 弘之, 松永 浩子, 内田 憲孝

    Patent

    J-GLOBAL

  • 大規模並列核酸分析方法

    松永 浩子, 神原 秀記

    Patent

    J-GLOBAL

  • 核酸の均一増幅方法および高感度検出方法

    神原 秀記, 松永 浩子, 谷口 妃代美

    Patent

    J-GLOBAL

  • 核酸の均一増幅方法および高感度検出方法

    特許第5519304号

    神原 秀記, 松永 浩子, 谷口 妃代美

    Patent

    J-GLOBAL

  • 大規模並列核酸分析方法

    松永 浩子, 神原 秀記, 梶山 智晴

    Patent

    J-GLOBAL

  • 大規模並列核酸分析方法

    松永 浩子, 神原 秀記, 梶山 智晴

    Patent

    J-GLOBAL

  • 核酸分析方法

    谷口 妃代美, 永井 啓一, 松永 浩子

    Patent

    J-GLOBAL

  • 遺伝子検査方法

    永井 啓一, 岡野 和宣, 野田 英之, 松永 浩子, 谷口 妃代美, 矢澤 義昭, 梶山 智晴

    Patent

    J-GLOBAL

  • 遺伝子検査方法

    特許第4122317号

    永井 啓一, 岡野 和宣, 野田 英之, 松永 浩子, 谷口 妃代美, 矢澤 義昭, 梶山 智晴

    Patent

    J-GLOBAL

  • 肺癌用遺伝子マーカー

    三橋 将人, 神原 秀記, 松永 浩子, 川村 雅文

    Patent

    J-GLOBAL

  • 肺癌用遺伝子マーカー

    特許第4392163号

    三橋 将人, 神原 秀記, 松永 浩子, 川村 雅文

    Patent

    J-GLOBAL

  • 遺伝子検査装置及び遺伝子検査方法

    松永 浩子, 村川 克二, 岡野 和宣, 宮原 裕二

    Patent

    J-GLOBAL

  • 分取装置

    松永 浩子, 植松 千宗, 岡野 和宣

    Patent

    J-GLOBAL

  • ポリヌクレオチドプローブチップ及びポリヌクレオチド検出法

    岡野 和宣, 神原 秀記, 植松 千宗, 松永 浩子, 入江 隆史, 梶山 智晴, 安田 賢二

    Patent

    J-GLOBAL

  • プローブチップ、プローブチップ作成方法、試料検出方法、及び試料検出装置

    特許第3829491号

    岡野 和宣, 神原 秀記, 植松 千宗, 松永 浩子, 入江 隆史, 梶山 智晴, 安田 賢二

    Patent

    J-GLOBAL

  • 核酸配列比較分析方法及び試薬キット

    松永 浩子, 岡野 和宣

    Patent

    J-GLOBAL

  • DNA塩基配列決定用試料作製方法及び試薬キット

    松永 浩子, 神原 秀記

    Patent

    J-GLOBAL

  • DNA解析方法及び試薬キット

    松永 浩子, 岡野 和宣, 神原 秀記

    Patent

    J-GLOBAL

▼display all