International Symposium on Bio-imaging andGene Targeting Sciences in Okayama
Contents
Greeting … ………………………………………………………………………………… p. 3
Location … ………………………………………………………………………………… p. 4
Program … ………………………………………………………………………………… p. 7
Abstract … ………………………………………………………………………………… p. 9

Date:
Sunday, February 15, 2015
Venue:The 50th Anniversary Hall, Okayama University(1-1-1 Tsushima-naka, Kita-ku, Okayama, 700-8530)
Organizers:
Okayama University (President: Prof. Kiyoshi Morita)
Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical
Sciences (Dean: Prof. Mitsune Tanimoto)
Okayama University Dental School (Dean: Prof. Takuo Kuboki)
Co-Organizer:
RIKEN, Institute of Physical and Chemical Research
Supporter:
Japan Science and Technology Agency (JST)Program Committee:
Satoshi Kubota (Chair)Toshitaka Oohashi
Hiroshi Kamioka
Takuya MatsumotoThis symposium is supported by a trust fund for Molecular Imaging Research
Strategies by MEXT-Japan, titled “Center of excellence for molecular imaging
specialists education in Okayama”, and a Grant-in-Aid for the COE projects by
MEXT-Japan, titled “Center of excellence for molecular and gene targeting therapies
with micro-doze molecular imaging modalities.”文部科学省科学技術試験研究委託事業
分子イメージング研究戦略推進プログラム

岡山分子イメージング高度専門人材育成事業
総括国際シンポジウム目次

拶… ………………………………………………………………………………… …p.…3

挨会 場 案 内… ………………………………………………………………………………… …p.…4
プログラム……………………………………………………………………………………… …p.…7
録……………………………………………………………………………………… …p.…9

抄【開 催 日】…

平成 27 年 2 月 15 日（日）

【会

場】

…

岡山大学創立五十周年記念館…

（〒700−8530【主岡山市北区津島中 1−1−1）

催】…

国立大学法人岡山大学（学長：森田…

岡山大学大学院医歯薬学総合研究科（研究科長：谷本光音）…

岡山大学歯学部（学部長：窪木拓男）

【共
…

催】
独立行政法人理化学研究所

【後
…

潔）

援】
独立行政法人科学技術振興機構【実行委員】…

久保田聡（委員長）

…

大橋俊孝

…

上岡

…

松本卓也

寛

このシンポジウムは，文部科学省科学技術試験研究委託事業 分子イメージング研究戦略推進プログ
ラム「岡山分子イメージング高度専門人材育成事業」，および，文部科学省概算要求特別経費（プロジェ
クト分）－国際的に卓越した教育研究拠点機能の充実－「分子イメージング・マイクロドーズ（第０相）臨床試験体制を擁する分子標的治療研究・教育拠点の構築－（独）理化学研究所との連携による教育研究基盤の確立－」事業の一環として開催されます．

International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama

Welcome to the International Symposium on Bio-imaging andGene Targeting Sciences in Okayama, announcing our current
achievement and future prospects on life sciences
Prof. Mitsune Tanimoto
Dean
Okayama University Graduate School of Medicine,
Dentistry and Pharmaceutical Sciences

Prof. Takuo Kuboki
Dean
Okayama University Dental School
On behalf of the organizer and program committee, we hereby sincerely welcome you to
our Okayama University. Based on the long tradition as a medical school, Okayama University
graduate School of Medicine, Dentistry and Pharmaceutical Sciences was born in 2005. Since
then, this Graduate School has fostered advanced medical care and advanced research,which plays an active role not only in Chugoku-Shikoku region, but also in Japan towards theworld.
Under the support of the Ministry of Education, Culture, Sports, Science and TechnologyJapan (MEXT), Okayama University has developed an educational project entitled the Program
for the Development of Highly Specialized Professionals on Bio-Imaging Medicine since 2011.
In order to conclude this grand project, the final symposium is going to be held under thetitle of International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama
in February, 2015. Key note lectures will be given by Dr. Takanori Saido, Senior Team Leader
in RIKEN Brain Science Institute and Prof. Eiji Matsuura, Vice Director of OMIC in Okayama
University. In these lectures, current knowledge on the aging process of neurons leading to
Alzheimer’s disease and novel therapeutic strategies against atherosclerosis and cancers,which are being explored in our OMIC projects will be presented. Furthermore, 3 symposium
sessions will introduce research frontiers that critically represent the direction for our future life
sciences through interdisciplinary research collaborations. In these sessions, leading scientists
in the world will be invited and will present hot topics on Nuclear Architecture Imaging，Nanobioengineering; and Bone and Cartilage Molecular Imaging, followed by active scientificdiscussion. We hope this symposium is enjoyable, and fruitful for all of the attendees in
exploring the life sciences for the next generation.

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International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama

Location
The 50th Anniversary Hall, Okayama University(1-1-1 Tsushima-naka, Kita-ku, Okayama, 700-8530)

The 50th Anniversary Hall

4

International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama

The 50th Anniversary Hall Layout*Complementary lunch boxes will be served in the 2nd-floor hallway (★).
5

International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama

Time Table

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International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama

ProgramSunday, February 15, 2015
9:00 - 9:10 a.m.

Opening Remarks

9:10 - 10:30 a.m.

Special Lectures

Special Lecture 1

Chair: Prof. Hideki Matsui (Okayama University)“Stop Preclinical Alzheimer’s Disease!”
Dr. Takaomi C. Saido (RIKEN Brain Science Institute)
Special Lecture 2…… p.10Chair: Prof. Takuo Kuboki (Okayama University)“Okayama Medical Innovation Center (OMIC)
and Molecular Targeting Technology”
Dr. Eiji Matsuura (OMIC, Okayama University)
10:30 a.m.

…… p.11Break

10:40 a.m. - 12:10 p.m.
Symposium -Session 1-

Chair: Prof. Satoshi Kubota (Okayama University)

Characterization of Cellular Status by Nuclear
Architecture Imaging
1. “Mechanisms of Heterochromatin Positioning
in the Nucleus”
Dr. Irina Solovei (Ludwig Maximilians University of Münich (LMU))…… p.16

2. “Role of Spatial Positioning of Chromosome Territories:
Evolutionary Views and Characteristics in Cancer Cells”
Dr. Hideyuki Tanabe (The Graduate University for Advanced Studies (SOKENDAI))…… p.18

3. “Visualization of Dynamics of Methylated DNA
in living cell and animal”
Dr. Kazuo Yamagata (Osaka University)
12:10 p.m.

…… p.20

Break*Complementary lunch boxes will be servedin the 2nd-floor hallway

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International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama
12:20 - 12:50 p.m.

Luncheon Seminar

Chair: Prof. Masaharu Takigawa (Okayama University)“Single Cell Transcriptome Analysis Dissects Cell Fate
Determination from iPS Cells to Cardiomyocyte”
Dr. Akira Watanabe (CiRA, Kyoto University)
12:50 p.m.

Break

1:00 - 2:30 p.m.

Symposium -Session 2-…… p.14Chair: Prof. Takuya Matsumoto (Okayama University)

Current Topics in Nano-Bioengineering
1. “Imaging and Gene Expression Patterning with
Micro- and Nanofluidics”
Dr. Shu Takayama (University of Michigan)…… p.21

2. “Nanobiomaterials for Diagnosis and Treatmentof Vascular Disease”
Dr. Hyun Joon Kong (University of Illinois)…… p.22

3. “Genetically-encoded Tools to Optically Control
and Image Ca2+ Dynamics”
Dr. Takeharu Nagai (Osaka University)
2:30 - 2:45 p.m.

Break

2:45 - 4:45 p.m.

Symposium -Session 3-…… p.23

Chair: Prof. Toshitaka Oohashi (Okayama University)
Prof. Hiroshi Kamioka (Okayama University)

Hot Topics in Bone and Cartilage Imaging
1. “Details in the Nano-world: Assessing Structure-function
Relationship of Cartilage by Atomic Force Microscopy”
Dr. Attila Aszodi (Clinical Center University of Münich (LMU))…… p.24

2. “A Feasible Study of Molecular Bio-imaging
of Articular Cartilage Proteoglycans”
Dr. Toshitaka Oohashi (Okayama University)…… p.26

3. “Quantitative Illumination on Bone Histology
and Cell Biology by Fluorescence Imaging”
Dr. Ji-Won Lee (Ehime University)…… p.28

4. “Bioimaging of Osteocytes in vivo and in vitro”
Dr. Hiroshi Kamioka (Okayama University)

4:45 - 5:00 p.m.

8

Closing Remarks…… p.29

Special Lectures

International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama
9:10 - 9:50 a.m.

Special Lecture 1

Chair: Prof. Hideki Matsui (Okayama University)

Stop Preclinical Alzheimer’s Disease!

Dr. Takaomi C. Saido
RIKEN Brain Science InstituteDeposition of amyloid ß peptide (Aß), the primary cause of Alzheimer’s disease (AD), in thebrain precedes the disease onset approximately by 25 years. This silent state, accompaniedby Aß pathology without the major symptoms, is referred to as preclinical AD. Because one
3rd of Japanese population is already over the age of 65, approximately one 3rd of Japanesepeople are in the state of preclinical AD. To minimize the number of patients and thus of caregivers, it is necessary to establish diagnosis/prognosis and prevention of AD in the preclinicalstate. Based on our achievements on metabolism of Aß and on generation of the most relevantmouse AD models, I will describe the present status and future perspective of AD research.

Academic Career:2009 – present

Visiting Professor in Japan Women’s College

2008 – presentGroup Director in RIKEN Brain Science Institute2006 – present

Visiting Professor in Graduate School of Agricultural and Life Sciences

2005 – present

Visiting Professor in Institute for Frontier Medical Sciences, University of Kyoto

2004 – present

Visiting Professor in University of Nagoya School of Medicine

1999 – 2000

Visiting Professor in University of Tsukuba School of Medicine

1997 – present

Visiting Professor in Tohoku University School of Medicine

1997 – present

Visiting Professor in Yokohama City Medical School

1997 – present

Laboratory Head in RIKEN Brain Science Institute

Visiting Professor in Waseda University

1992

Visiting Scientist in Scripps Institute1988 – 1997

Research Scientist in Tokyo Metropolitan Institute of Medical Science

10International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama
9:50 - 10:30 a.m.

Special Lecture 2

Chair: Prof. Takuo Kuboki (Okayama University)

Okayama Medical Innovation Center (OMIC)
and Molecular Targeting Technology

Dr. Eiji Matsuura
Collaborative Research Center for OMIC, and Department of Cell Chemistry,
Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences

Okayama Medical Innovation Center (OMIC), which has fully equipped molecular imaging
research facilities, was established at Okayama University (Medical Campus) as a collaborative
research center for industry-academia-government teams in 2009 by the Japanese Scienceand Technology Agency (JST) with the aim of revitalizing regional industries and started
operating since April, 2011. In addition, the MEXT Project for Creation of Research Platforms
and Sharing of Advanced Research Infrastructure promotes the joint usage (of OMIC) of
advanced research facilities and equipment possessed by universities and independent
research institutes for industry, academia, and government organizations (since 2013). The
project could contribute to achieving vital issues through technological innovation. The center
also assists educational programs on molecular imaging researches at the Okayama University
Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, in corporation with
Riken (Kobe).
Up to date, we have been promoting several original research programs supported by JST,
MEXT, and other governmental grants at the center. In those studies, we are now establishinga novel molecular targeting technology, namely, Theranostics (Thera- Diagnostics), of which
concept is that the combination technology simultaneously providing both of “Therapeutic”
and “Diagnostic” effects to the patients with cancer, atherosclerotic, and other diseases. For
the technology, we can utilize humanized and a shorten antibody variant (single chain Fv;
scFv) that is labeled with 64Cu or 89Zr (a novel PET nuclide), and a drug delivery system (DDS)
consisted of novel bio-degradable polymers.
In the lecture, I would like to introduce the OMIC molecular imaging center and to overview
our ongoing research projects.

Academic Career:
2011 – present

Professor, Collaborative Research Center for OMIC, and Department of Cell Chemistry, Okayama University Graduate
School of Medicine, Dentistry and Pharmaceutical Sciences

2004 – 2011

Associate Professor, Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry andPharmaceutical Sciences

2001 – 2004

Associate Professor, Department of Cell Chemistry, Okayama University Graduate School of Medicine and Dentistry

1997 – 2001

Assistant Professor, Department of Cell Chemistry, Institute of Cellular and Molecular Biology, Okayama University
Medical School

1995 – 1997

Research Associate, Department of Biochemistry, Hokkaido University School of Medicine

1988 – 1995

Senior Research Scientist, Diagnostics Division, Yamasa Corporation

1986 – 1988

Research Associate, Department of Pediatrics/Medicine, National Jewish Center for Immunology and Respiratory
Medicine, Denver, CO, USA

1984 – 1986

Research Associate, Department of Immunochemistry, Faculty of Pharmaceutical Science, University of Okayama

11Luncheon Seminar

International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama
12:20 - 12:50 p.m.

Luncheon Seminar

Chair: Prof. Masaharu Takigawa (Okayama University)

Single Cell Transcriptome Analysis Dissects Cell Fate
Determination from iPS Cells to Cardiomyocyte

Dr. Akira Watanabe
Core Facility of Genome and Epigenome Analysis, Center for iPS Cell Research and Application (CiRA),
Kyoto University

During human development, a single-cell fertilized egg generates hundreds of different typesof the cell. This cell fate specification is finely regulated by epigenetic mechanism. However,there is little understanding of the mechanisms by which a particular gene network at a branchof the cell differentiation defines direction to specific cell type. For the clinical application, it
is also important to generate a specific cell type with a high efficiency, by precise control ofdifferentiation. Continuous supply of cardiomyocyte is required for cell-based therapy anddrug screening to evaluate cardiotoxicity. We aim to understand the detailed dynamics oftranscription during differentiation from induced pluripotent stem (iPS) cells to cardiomyocyte
and increase the efficiency of cardiac differentiation.
We performed single cell RNA-seq using iPS and cardiomyocyte-directed cells. We
differentiated human iPS cells into cardiomyocyte by changing the growth factors at eachtime point, and harvested the cells at day 1, 3, 5, 7, 9, 21 and 30 after induction of directeddifferentiation. Twenty-four of the singlet cells harvested at each time point were then applied
into C1 Single Cell Auto-Prep System for cDNA synthesis, followed by library preparation using
NexteraXT DNA Sample Prep Kit (Illumina). Massively parallel sequencing was performed
by HiSeq2500, and sequencing data was processed by mapping by Tophat and calculating
relative expression value RPKMforgenes. Principle component analysis showed heterogeneityof gene expression in each day point, and enabled data to sort samples into differentiation
status. Our newly developed method modified from Weighted Correlation Network Analysis(WGCNA) identified core gene expression modules of differentiated and iPS cells. In addition,
we could detect several groups of genes whose expression were dynamically changed during
the differentiation process. We propose time-information-free analysis as a powerful approachfor unveiling the dynamics of transcriptome in reprograming and differentiation.

Academic Career:2009 – present

Assistant Professor in the Core Facility of Genome and Epigenome Analysis, Center for iPS Cell Research andApplication (CiRA), Kyoto University

2003 – 2009

Postdoctoral fellow in Research Center for Advanced Science and Technology, the University of Tokyo

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SymposiumInternational Symposium on Bio-imaging and Gene Targeting Sciences in Okayama
10:40 a.m. - 12:10 p.m. Symposium -Session 1-

Chair: Prof. Satoshi Kubota (Okayama University)

Characterization of Cellular Status by Nuclear Architecture Imaging
1. Mechanisms of Heterochromatin Positioning
in the Nucleus

Dr. Irina Solovei
Human Biology and Bioimaging group,
Ludwig Maximilians University of Münich (LMU)

Spatial segregation of transcriptionally active euchromatin and silent heterochromatin
is an important factor regulating nuclear functions. Majority of the eukaryotic nuclei have
conventional architecture with transcriptionally active euchromatin residing in the nuclear
interior and heterochromatin abutting the nuclear periphery and the nucleolus. Recently wefound a unique exception from the above rule, nuclei of rod photoreceptor cells of nocturnal
mammals. For optical reasons, heterochromatin is concentrated in the center of these nuclei
whereas euchromatin lines the nuclear periphery, thereby forming an inverted nuclear
organization in comparison to conventional nuclei. In both conventional and inverted nuclei,
chromosomes acquire a complex folded structure which adapts to the shape of the nucleus
and secures correct intranuclear positioning of eu- and heterochromatin regions.
To elucidate possible mechanisms of establishing of inverted versus conventional nuclear
architecture, we carried out a detailed study of epigenetic landscape in both nuclear types. We
showed that major epigenetic factors associated with eu- or heterochromatin remain similar inconventional and inverted nuclei. Moreover, depletion of methylation code writers (e.g., Suv39, Suv4-20, G9a) or readers (e.g., MECP2) does not affect global nuclear architecture in bothcases.
Next we analyzed spatial arrangement of heterochromatin in tissues from wild type andmice with mutations in the lamin B receptor (Lbr) and lamin A/C (Lmna) genes. We identified
two mechanisms tethering peripheral heterochromatin to the nuclear envelope, an LBRdependent and lamin A/C-dependent, which are sequentially used at early and late stagesof differentiation, respectively. Tethers have opposite effects on the expression of tissuespecific genes: selective disruption of lamin A/C downregulates whereas absence of LBR
upregulates muscle gene expression. Importantly, the absence of both LBR and LA/C leads toloss of peripheral heterochromatin and inversion of nuclear architecture with heterochromatin
localizing to the nuclear interior in non-rod cells.
Taken together, our data suggest that the major epigenetic factors do not play a crucialrole in the choice between inverted and conventional nuclear architecture. Conventional
mammalian nuclei relay on strong redundancy of epigenetic code itself and its writers, whereas
the inversion in rods relies on absence of specific readers, LBR- and lamin A/C-dependent
peripheral heterochromatin tethers.

16International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama
Academic Career:2009 – present

Principle Investigator in Ludwig Maximilians University of Munich (LMU), Human Biology and Bioimaging group

1996 – 2009

Senior Research Scientist in Ludwig Maximilians University of Munich (LMU), laboratory of Prof.T.Cremer

1990 – 1996

Senior Research Scientist in the Biological Research Institute of the St-Petersburg University and Wellcome Trust
Fellow in University of Leicester (UK), laboratory of Prof.H.Macgregor

1984 – 1990

Research Scientist in the Biological Research Institute of the St-Petersburg University

1984

Doctor Degree of Biological Science (PhD) from Department of Cell Biology and Histology, Biological Faculty,
University of St-Petersburg

17International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama
10:40 a.m. - 12:10 p.m. Symposium -Session 1-

Chair: Prof. Satoshi Kubota (Okayama University)

Characterization of Cellular Status by Nuclear Architecture Imaging
2. Role of Spatial Positioning of Chromosome Territories:
Evolutionary Views and Characteristics in Cancer Cells

Dr. Hideyuki Tanabe
Department of Evolutionary Studies of Biosystems, School of Advanced Sciences,
The Graduate University for Advanced Studies (SOKENDAI)

Chromosomes are discretely, highly compartmentalized within the cell nucleus in eukaryotes
forming so-called “chromosome territories (CTs)”. It has been studied for nearly two decades,
firstly in humans, other mammals, and chickens by utilizing 3D-FISH techniques. How do CTs
occupy the cell nucleus? What kind of regulations can be applied to arrange their positioning?
From previous studies for this decade, it has been revealed that radial positioning of CTs
from center to nuclear rim has the following characteristics. 1) Radial positioning of CTs
depends on the physical size and gene density of each CT; larger gene-poor CTs are locatedtoward periphery and smaller gene-dense CTs are located into interior of the nucleus. For
example, human 18 and 19 CTs are gene poor and gene dense, respectively, which localize
in periphery or in interior regions discretely in human lymphocytes. 2) Evolutionarily syntenic
regions of CTs are inclined to localize the same radial positioning among species. It has
been demonstrated that the topology of syntenic CTs with human 18 and 19 chromosomes is
evolutionarily conserved in chickens as well as in primates. Especially, gibbon syntenic CTs
with human 18 and 19 chromosomes are divided into several pieces but their radial positioningis highly conserved. 3) Radial positioning of CTs depends on the region of Lamin AssociatedDomains (LADs); LADs are located near the nuclear rim, which are tethering mainly gene-poor
chromosomal regions corresponding to G/C-bands roughly consisting of heterochromatin.
4) Actin related protein 6 (Arp6), which is one of the ubiquitous components of chromatin
remodeling complexes conserved from yeast to human, has affected to global nuclear radial
distribution of CTs. Arp6-knock out chicken DT40 cells have shown disturbed global nuclear
architecture. 5) Cancer cells have shown almost same characteristics with normal cells,
however, in glioblastoma cells radial positioning of CTs has been disturbed intensely revealedby peripheral versus interior localizing combined CTs as probes for 3D-FISH techniques.
Collectively, spatial radial positioning of CTs could be affected strongly with physicalproperties such as gene density, LADs association, and Arp6 related mechanisms, whereas
less affected with the status of gene expression or epigenetic factors. Organization of nuclear
architecture from evolutionary views will be discussed.

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International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama
Academic Career:
2006 – present

Associate Professor in Department of Evolutionary Studies of Biosystems, School of Advanced Sciences, The
Graduate University for Advanced Studies (SOKENDAI)

2003 – 2006

Associate Professor in Department of Biosystems, School of Advanced Sciences, The Graduate University for
Advanced Studies (Sokendai)

2001 – 2003

Senior Staff Scientist in Division of Genetics and Mutagenesis, National Institute of Health Sciences

1999 – 2001

Long Overseas Scientist (STA fellow): Prof. Thomas Cremer’s laboratory, Institute of Anthropology and Human
Genetics, Ludwig Maximilians University, Münich, Germany

1998

Doctor Degree of Science (Thesis Doctor) from Graduate School of Science, Hokkaido University

1993 – 1998

Staff Scientist: JCRB Cell bank, Division of Genetics and Mutagenesis, National Institute of Health Sciences

1991

Master Degree of Science from Graduate School of Science, The University of Tokyo

1989

Graduation from Department of Anthropology, Faculty of Science, The University of Tokyo

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International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama
10:40 a.m. - 12:10 p.m. Symposium -Session 1-

Chair: Prof. Satoshi Kubota (Okayama University)

Characterization of Cellular Status by Nuclear Architecture Imaging
3. Visualization of Dynamics of Methylated DNA
in living cell and animal

Dr. Kazuo Yamagata
Research Institute for Microbial Diseases,
Osaka University

In mammals, DNA is methylated at CpG sites, which play pivotal roles in gene silencing
and chromatin organization. Furthermore, DNA methylation undergoes dynamic changes
during development, differentiation, and in pathological processes. The conventional methods
represent snapshots; therefore, the dynamics of this marker within living organisms remains
unclear. To track this dynamics, we made a knockin mouse that expresses a red fluorescentprotein (RFP)-fused methyl-CpG-binding domain (MBD) protein from the ROSA26 locus
ubiquitously; we named it MethylRO (methylation probe in ROSA26 locus). Using this mouse,
we performed RFP-mediated methylated DNA immunoprecipitation sequencing (MeDIP-seq),
whole-body section analysis, and live-cell imaging. We discovered that mobility and pattern ofheterochromatin as well as DNA methylation signal intensity inside the nuclei can be markers
for cellular differentiation status. Thus, the MethylRO mouse represents a powerful bioresource
and technique for DNA methylation dynamics studies in developmental biology, stem cellbiology, as well as in disease states.

Academic Career:2010 – present

Associate professor in Research Institute for Microbial Diseases, Osaka University

2007 – 2010

Research scientist in RIKEN Center for Developmental Biology

2003 – 2007

Assistant professor in Graduate School of Life and Environmental Sciences, University of Tsukuba

2000 – 2002

JSPS postdoctoral research fellow at Genome Information Research Center, Osaka University

2000

Ph.D. from University of Tsukuba Graduate School of Agriculture

20International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama
1:00 - 2:30 p.m. Symposium -Session 2-

Chair: Prof. Takuya Matsumoto (Okayama University)

Current Topics in Nano-bioengineering
1. Imaging and Gene Expression Patterningwith Micro- and Nanofluidics

Dr. Shu Takayama
Biomed Eng and Macromolecular Science & Engineering,
University of Michigan

This presentation will present work from our laboratory that combines micro- and nanofluidics
with imaging and gene expression. The specific nanofluidic technology to be described
include use of fracture-fabricated tunable nanochannels for single chromatin fiber linearizationand multi-color imaging of histone modifications along the fiber. In microfluidic topics, I will
describe efforts in our lab in studying GPCR signaling in response to computer-controlled
microfluidic pulsed stimulation. Live cell imaging of cells with genetically engineered protein
reporters and analysis of the cell response with computer models of the signaling pathway
enable non-invasive dissection of cell signaling pathways. We also demonstrate that there is an
optimal stimulation frequency at which transcription factor activation is maximized and is larger
than can be obtained with continuous stimulation. Finally, use of aqueous two phase system
micropatterning to perform patterned gene expression and knockdown of genes in selectregions of a monolayer of cells with be described. Time permitting some of our latest results in
microfluidic in vitro fertilization studies will be presented.

Academic Career:2013 – present

Associate Director in Michigan Center for Integrative Research in Critical Care2013 – present

Director in NIH Microfluidics in Biomedical Sciences Training Program

2010 – present

Professor, Biomed Eng and Macromolecular Science & Engineering, U. of Michigan

2010 – present

WCU (till 2013) then Adjunct (from 2013) Professor, UNIST, Korea

2010 – 2014

Associate Chair for Translational Research, Biomed Eng Dept, U. of Michigan

2006 – 2010

Associate Professor, Biomed Eng and Macromol Sci. & Eng., U. of M.

2000 – 2005

Assist Professor, Biomed Eng and Macromol Sci. & Eng., U. of M.

1998 – 2000

Leukemia and Lymphoma Society Postdoctoral Fellow, Harvard University

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International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama
1:00 - 2:30 p.m. Symposium -Session 2-

Chair: Prof. Takuya Matsumoto (Okayama University)

Current Topics in Nano-bioengineering
2. Nanobiomaterials for Diagnosis and Treatmentof Vascular DiseaseDr. Hyun Joon Kong
Departments of Chemical and Biomolecular Engineering, Bioengineering, Pathobiology, Center
for Biophysics, University of Illinois at Urbana-Champaign

The human body is highly vascularized to transport oxygen, nutrients, and hormones toand from cells residing in tissue and organ systems. A variety of intrinsic and extrinsic factors
can cause occlusion, leakage, or rupture of vasculature, thus leading to tissue ischemia,
necrosis, and ultimately death. To date, extensive efforts have been made to detect and treat
these vascular diseases with diverse bioactive molecules, imaging probes, and stem cells.
These diagnostic and therapeutic modalities are often integrated with various engineering
technologies to further improve their performance. In order to advance these efforts, we havedeveloped several implantable nanobiomaterial systems to elevate the quality of vascularimaging, repair and regeneration by integrating materials chemistry and characterization withbiotransport phenomena. In this talk, I will highlight a few of the diagnostic and treatment tools
we have developed, including (1) a gadolinium-coated nanoparticle designed to enhance thequality of magnetic resonance imaging (MRI) of ischemic tissue (Fig. 1a), (2) nanomaterials fordelivery of stem cells to inflamed blood vessels (Fig. 1b), (3) a “living” microvascular stamp
to control the organization of blood vessel during regeneration (Fig. 1c), and (4) a self-foldinghydrogel to modulate vascular drug release (Fig. 1d).

Fig.1 Materials for imaging and treatment of vascular diseases

Academic Career:2013 – present

Associate Professor, Departments of Chemical and Biomolecular Engineering, Bioengineering, Pathobiology, Center
for Biophysics, University of Illinois at Urbana-Champaign

2007 – 2013

Assistant Professor, Departments of Chemical and Biomolecular Engineering, Bioengineering, Pathobiology, Center
for Biophysics, University of Illinois at Urbana-Champaign

2004 – 2006

Research Associate, School of Eng. and Applied Sci. (Bioengineering) Harvard University

2001 – 2004

Post-Doctoral Research Fellow, Dept. of Biological and Materials Sci. Univ. of Michigan

1997 – 2001

Research Assistant, Dept. of Chemical Eng. & Civil & Environmental Eng. Univ. of Michigan

22International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama
1:00 - 2:30 p.m. Symposium -Session 2-

Chair: Prof. Takuya Matsumoto (Okayama University)

Current Topics in Nano-bioengineering
3. Genetically-encoded Tools to Optically Control
and Image Ca2+ Dynamics

Dr. Takeharu Nagai
The Institute of Scientific and Industrial Research,
Osaka University

In living organism, Ca2+ is one of the most versatile second messenger to control biologicalprocesses such as muscle contraction, hormonal secretion and apoptosis induction. Its
spatial and temporal dynamics has key roles to regulate these physiological phenomena.
To reveal such dynamics, variety of Ca 2+ indicators had been developed. They enabled
noninvasive visualization of Ca2+ dynamics, provided meaningful information for research inwide range of biological field. However, for deeper understanding of relationship between thespatiotemporal Ca2+ dynamics and the following response, development of tools to manipulate
intracellular Ca2+ level have been desired. In current methods, Ca2+ concentration can becontrolled by light through Ca2+ binding compounds with photocleavable moieties. However,they require irradiation of toxic ultraviolet wavelength light and/or cell loading associatedwith disruption of the cell membrane. These properties which have possibility to impair cells
become big problem especially in the case of in vivo measurement. In addition to this, Ca2+
release from such compounds is irreversible. To overcome this, we developed a geneticallyencoded photoactivatable Ca2+ releaser called PACR (PhotoActivatable Ca2+ Releaser). Thatis composed of Ca2+ binding protein and light-sensitive protein. Affinity of PACR for Ca 2+
was decreased during irradiation of blue light. Thus reversible and repeatable increasingof Ca 2+ concentration in cell is possible without damage to living specimens. By using
PACR, we succeeded nucleus specific temporal Ca2+ increase in HeLa cells and excitationof specific neuron in freely moving C. elegans by blue light irradiation. This useful tool is
expected to contribute on researches to reveal the role of Ca2+ dynamics in complex biologicalphenomena. In addition to this manipulation tool, I would like to introduce color variants ofsuper-duper luminescent protein that we developed recently, which can be used compatiblywith optogenetic actuators.

Academic Career:2014 – present

Vice Director, The Institute of Scientific and Industrial Research, Osaka University

2012 – present

Visiting senior chief researcher, Quantitative Biology Center, RIKEN

2012 – present

Professor, The Institute of Scientific and Industrial Research, Osaka University

2008 – 2014

Researcher, PRESTO, JST2005 – 2012

Professor, Research Institute for Electronic Science, Hokkaido University

2001 – 2005

Researcher, PRESTO, JST

1998 – 2001

Researcher, RIKEN

1995 – 1998

Research fellow, JSPS

23

International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama
2:45 - 4:45 p.m.

Symposium -Session 3-

Chair: Prof. Toshitaka Oohashi (Okayama University)
Prof. Hiroshi Kamioka (Okayama University)

Hot Topics in Bone and Cartilage Imaging
1. Details in the Nano-world: Assessing Structure-function
Relationship of Cartilage by Atomic Force Microscopy

Dr. Attila Aszodi
Experimental Surgery and Regenerative Medicine, Department of Surgery,
Clinical Center University of Münich (LMU)

The performance of most tissues in vertebrates crucially depends on their structuralmechanical properties, which is determined by matrix-matrix, cell-matrix and cell-cellinteractions. Connective tissues of the skeletal system accommodate for recurring mechanicalstress by building up hierarchical macromolecular structures that provide biomechanicalstability. Cartilage is a macromolecular fiber (collagen)/gel (proteoglycan) composite material
which withstands compressive, shear and tensile forces. The growth plate (GP) and thearticular cartilage (AC) are specialized structures which drives the longitudinal elongation of
the skeletal elements and dissipate load in joints, respectively. Mutations in genes coding for
extracellular matrix proteins and their receptors often affect the organization of these structures
leading to skeletal pathologies such as chondrodysplasia and osteoarthritis.
Atomic force microscopy (AFM) offers a unique opportunity to simultaneously assess the
structural and mechanical properties of biological tissues in their physiological environment.
AFM operated either on the native surface or cryo-sections of cartilage provides a nondestructive way for quantification of morphological and mechanical data at the nanometerscale. Recently, we have applied AFM-based imaging and nano-indentation (IT) techniques tocharacterize structural and biomechanical properties of the developing, normal and diseasedmouse cartilaginous tissues. Our data implicates that nanoscale IT-AFM is a sensitive tool tomonitor structural and functional properties of the mouse GP and AC in growing endochondralbones and to distinguish between healthy and diseased cartilage. AFM-imaging is capable
to capture overview and ultrastructural details on tissue slices. IT-AFM measurements enable
the analysis of matrix components such as collagen II fibrils or the proteoglycan moiety. The
combination of AFM imaging and indentation measurements demonstrates that a decreasein fibril density is accompanied by reduced nano-stiffness of the proteoglycan gel likely due
to lower osmotic swelling pressure exerted by the composite material. Thus, the structural
architecture can be directly correlated with the compressive stiffness of various matrix
compartments at the level of collagen fibrils and proteoglycans. IT-AFM measurementsof chondrodysplastic mutant mouse strains suggest that the main cartilage matrix protein
collagen II and its ß1 integrin receptors have a pivotal role in the control of morphogenesis andin the regulation of the mechanical properties of the GP. The dramatic reduction of the elastic
modulus in Col2a1-null GP cartilage evidences that collagen II is the major protein which
determines the compressive stiffness of the cartilage. ß1 integrins, besides playing a key role24International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama
for proliferative chondrocytes to adopt their elongated shape and columnar arrangement, also
modulate cartilage biomechanics likely via the control of collagen matrix assembly.

Academic Career:
2011 – present

Chair, Group leader, Experimental Surgery and Regenerative Medicine, Department of Surgery, Clinical Center
University of Münich (LMU)

2002 – 2010

Group leader, Max Planck Institute of Biochemistry, Department of Molecular Medicine, Martinsried, Germany

1998 – 2001

Assistant professor, Department of Experimental Pathology, Lund University, Lund, Sweden

1995 – 1997

Postdoctoral fellow, Department of Protein Chemistry, Max-Planck Institute for Biochemistry, Martinsried, Germany

1990 – 1994

PhD student, Agricultural Biotechnology Center, Gödöllö, Hungary

1988 – 1989

Scientific co-worker, Institute for Drug Research, Budapest, Hungary

25International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama
2:45 - 4:45 p.m.

Symposium -Session 3-

Chair: Prof. Toshitaka Oohashi (Okayama University)
Prof. Hiroshi Kamioka (Okayama University)

Hot Topics in Bone and Cartilage Imaging
2. A Feasible Study of Molecular Bio-imaging
of Articular Cartilage Proteoglycans

Dr. Toshitaka Oohashi
Department of Molecular Biology and Biochemistry,
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University

In the process of cartilage degeneration seen in osteoarthritis, loss of proteoglycan fromarticular cartilage has been widely accepted as a critical early event, followed by collagendegradation designated as a point of no return. Loss of articular cartilage in osteoarthritis isindirectly evaluated by radiography as a joint space narrowing in the most common diagnosis,
but early lesions of cartilage damage cannot be detected. Although several negatively charged
contrast agents were employed, they provided indirect images of the articular cartilage basedon the electrostatic repulsion with anionic glycosaminoglycans (GAGs) in the extracellularmatrices (ECMs). Recent advance in the development of targeted molecular probes and newimaging modalities enabled the detection of qualitative and functional change of articularcartilage. In this session, we report the recent progress of bio-molecular imaging of articularcartilage including our molecular probes targeting articular GAGs.
Chondroitin sulfate, a major component of articular cartilage, has negative charge causedby sulfates. Thus we designed lysine oligomers (monomer - pentamer) which were connectedwith the a-carboxylic acid and e-amino group. These lysine oligomers possess a-amino groups
as cationic moieties. NBD was used as a fluorescent group. Triiodobenzene (TIB) was used
for a X-ray contrast medium. Estimation of the length between periodic sulfates in chondroitin
sulfates using X-ray data predicted that lysine tetramer derivative Kε4-NBD and lysine
pentamer derivative Kε5-NBD possess potent affinity to chondroitin sulfates. Evaluation of theaffinity of the lysine oligomers to chondroitin sulfate by fluorescence polarization showed that
Kε5-NBD possesses most potent affinity to chondroitin sulfates of all. In addition, Kε4 andKε5-NBD stained articular cartilage ECM efficiently. Then, we created a novel articular cartilage
imaging X-ray probe Kε4-TIB and demonstrated an ex vivo imaging of articular cartilage in rat
osteoarthritic models. The imaging could quantitate loss of proteoglycan from osteoarthritic
cartilage.
Kε4-TIB may have a potential of in vivo X-ray imaging in animal arthritic models, which
contribute to drug discovery research for osteoarthritis.

26

International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama
Academic Career:2014 – present

Professor, Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Dentistry andPharmaceutical Sciences, Okayama University

2005 – 2014

Associate Professor, Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Dentistry andPharmaceutical Sciences, Okayama University

1998 – 2005

Lecturer, Department of Molecular Biology and Biochemistry, Okayama University Medical School

1996 – 1997

Research Fellow, in Max-Planck Institute of Biochemistry (Prof. Fässler), JSPS Postdoctoral Fellow for Research in
COE Abroad

1992 – 1997

Assistant Professor, Department of Molecular Biology and Biochemistry, Okayama University Medical School(Leave of absent: 1996-1997)27

International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama
2:45 - 4:45 p.m.

Symposium -Session 3-

Chair: Prof. Toshitaka Oohashi (Okayama University)
Prof. Hiroshi Kamioka (Okayama University)

Hot Topics in Bone and Cartilage Imaging
3. Quantitative Illumination on Bone Histology
and Cell Biology by Fluorescence Imaging

Dr. Ji-Won Lee
Division of Bio-Imaging, Proteo-Science Center (PROS),
Ehime University,

Fluorescence microscopy has revealed considerable detail in cellular structures andfunctions by specific labeling with a high resolution. This fluorescence imaging system isalso attractive tool as the complementation of the biochemical “quantitative” analysis andhistological “spatial” analysis in the cellular events. In this symposium, we will introduce about
our current applications of fluorescent imaging to bone research. We first observed spatially
distinct distributions of specific protein from osteocytic lacunae in rat femurs, demonstrating
the relative expression levels of these specific proteins exhibited reciprocally reversed patterns
in midshaft cortical bone through three-dimensional immunofluorescence morphometry andquantification. These observations guided us to investigate mechanistic study of cellular signal.
We next found the dynamic rearrangement of actin cytoskeletal affect to osteoclast functions
such as cell locomotion by using real-time imaging and super-resolution microscopy. Our recent
findings and several approaches for visualization and quantifying the cellular and molecularsignal suggest that fluorescence imaging enables us to distinguish the cellular functions fromgenetic mutations as well as satisfying with a much better resolution. Quantitative approachesof fluorescence imaging, as introduced here, will provide us further unprecedented insights inbone biology

Academic Career:2013 – present

Assistant Professor, Division of Bio-Imaging, Proteo-Science Center (PROS), Ehime University,

2011 – 2013

Post-doctoral fellowship (supported by Japan Society for the Promotion of Science), Department of Oral Pathology,
Tokyo Medical and Dental University

2009 – 2011

Post-doc fellowship, Research Institute for Biological Functions, Chubu University

2009 – 2010

Visiting Researcher, Section of Molecular Craniofacial Embryology, Tokyo Medical and Dental University

2008 – 2009

Visiting Researcher, Institute for Oral Science, Matsumoto Dental University

2008

Visiting lecturer, Institute for Oral Science, Matsumoto Dental University

2007 – 2008

Lecturer, Department of Food Science and Technology, Keimyung University – Microbiology

2006 – 2007

Lecturer, Department of Food Science and Technology, Daegu University – Microbiology

2005 – 2007

Lecturer, Department of Food Science and Technology, YoungNam College – Biochemistry, Food Processing andpreservation

2002 – 2008

Researcher, TMR (Traditional Microorganism Research) center, Keimyung University

28

International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama
2:45 - 4:45 p.m.

Symposium -Session 3-

Chair: Prof. Toshitaka Oohashi (Okayama University)
Prof. Hiroshi Kamioka (Okayama University)

Hot Topics in Bone and Cartilage Imaging
4. Bioimaging of Osteocytes in vivo and in vitro

Dr. Hiroshi Kamioka
Department of Orthodontics,
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University

In a variety of scientific fields, it is a worthwhile topic to visualize natural phenomenon. Newly
developed visualizing method often leads breakthrough in the scientific fields. Especially, inthe biological field, it is significant to reveal temporal-spatial response happened in the cellswith visualizing molecular level phenomenon. Such visualization could provide information
to understand cellular behavior to their extracellular stimulus in vivo and in vitro. Althoughosteocytes are the most abundant cells in bone, it has been difficult to study their biologicalfeature because they are embedded in hard bone tissue. So, even the real 3D structure of
the osteocyte was not uncovered till lately. On the other hand, newly developed techniqueof visualization was recently introduced in the bone cell biology. In this presentation, we
will introduce our application of confocal laser scanning microscopy to visualize osteocyte
morphology in bone, calcium imaging of osteocyte in vivo/in vitro to show real time responseof osteocytes, and the combination of ultra-high voltage electron microscopy and computer
simulation of fluid flow to reveal mechanosensitivity of osteocytes in bone.

Academic Career:2014 – present

Professor in Dept. of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama
University

2011 – 2015

Visiting Lecturer in Dept. of Biomechanics, Research Center for Nano Medical Engineering, Institute for Frontier
Medical Sciences, Kyoto University

2005 – 2013

Associate Professor in Dept. of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,
Okayama University

1999 – 2005

Lecturer in Dept. of Orthodontics, Okayama University Hospital(1995 – 1998)

Post-doctral fellow in Dept. of Anatomy, Indiana University Medical School

1993 – 1999

Assistant Professor in Dept. of Orthodontics, Tokushima University Dental School29

International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama

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