CiRA Annual Report2010 - 京都大学iPS細胞研究所 CiRA（サイラ）

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CiRA
A N N U A L
R E P O R T
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CiR A A N N U A L
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Message from the CiRA Director
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Organization Chart
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Research Groups
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Department of Reprogramming Science
34 Department of Clinical Application
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Shinya Yamanaka
34
Tatsutoshi Nakahata
10
Yasuhiro Yamada
36
Haruhisa Inoue
12
Yoshinori Yoshida
38
Hidetoshi Sakurai
14
Masato Nakagawa
16
Keisuke Okita
40 Department of Regulatory Science
18
Kazutoshi Takahashi
40
Takafumi Kimura
20
Knut Woltjen
42
Takashi Aoi
22
Takuya Yamamoto
44
Isao Asaka
24
Akitsu Hotta
26
Department of Cell Growth and Differentiation
26
Junya Toguchida
28
Jun Takahashi
30
Jun K.Yamashita
32
Kenji Osafune
2
46
ResearchProjects
48
IntellectualProperty
50
PickupfromPressRelease
51
HonorsandAwards
52
CiRAintheMedia
54
Events
58
Publications
59
Operation
60
iPSCellResearchFund
62
Glossary
64
CiRAFacilities
SmoothMuscleCellsand
StriatedMuscleCells
NeuronalCells
HumaniPSCells
SmoothMuscleCells
NeuronalCells
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Message from the CiRA Director
On April 1, 2010, Kyoto University restructured its Institute for
Integrated Cell-Material Sciences (iCeMS) to establish the Center
for iPS Cell Research and Application (CiRA), as a research center
focused on the study of induced pluripotent stem (iPS) cells. At
present, CiRA is home to around 200 research faculty and students
working to realize the clinical promise of these cells at the earliest
possible moment.
CiRA has 19 principal investigators working in research divisions of
Reprogramming Science, Cell Growth and Differentiation, Clinical
Application, and Regulatory Science. The research facility, completed
in February 2010, follows an open laboratory design and implements
an intensive seminar schedule to encourage interactivity and
opportunities for discussion among our scientists, and facilitate the
unfettered translation of basic science through preclinical research
and ultimately to clinical studies.
Note :ThisAnnualReportintroducesactivities
fortheperiodofApril1toDecember31,2010.
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In 2010, in addition to developing a system for supporting
effective research activity, we also achieved a number of
important results, including the development of a safer
method for iPS cell generation, preparations for safety
testing of iPS cell-based cell treatments in a primate
model of disease, and for the establishment of an iPS cell
bank. We are also now gearing up to study pathology and
drug screening using disease-specific iPS cell lines.
CiRA research has been conducted with support from the
Project for the Realization of Regenerative Medicine,
designed by the Ministry of Education, Culture, Sports,
Science and Technology, and other national initiatives. In
March 2010, the Cabinet Office established the iPS Cell
Project for Regenerative Medicine under its Funding
Program for World-Leading Innovative R&D on Science
and Technology (FIRST). This project will seek to develop
standardized methods for the generation of iPS cells, a
requisite for the realization of medical applications. We
also continue to collaborate with other institutions and
companies in Japan and abroad.
In addition to our scientific work, we have accelerated our
efforts to ensure that our iPS cell technologies are widely
disseminated. We have worked with a team of specialists
in Europe and the U.S. to work towards obtaining
international patents relating to iPS cell technologies as
part of our overall patent strategy. Additionally, we have
coordinated with bioresource distribution facilities to
promote widespread access to plasmids and other
materials needed to generate iPS cells. We also hold
multiple practical lectures and training courses to teach
techniques for iPS cell generation and culture.
Since the first demonstration of iPS cell generation in
mouse in 2006, we have seen a remarkable number of
advances in this field around the world, but many
challenges, including cell safety, methods for inducing
differentiation, transplantation protocols, and the
utilization of iPS cells in drug discovery and medicine, still
remain. We are striving through our research to bring the
promise of iPS cells to reality for many patients who see
these as a source of hope. In closing, I would like to offer
my sincere thanks to all the many people who have so
generously donated to the iPS Cell Research Fund, and
express my hopes for your continued support in the
future.
ShinyaYamanaka,M.D.,Ph.D.
Director,CenterforiPSCellResearchand
Application(CiRA),KyotoUniversity
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CenterforiPSCellResearchandApplication
KyotoUniversity(CiRA)
OrganizationChart
Director
AsofMarch1,2011
ShinyaYamanaka
Deputy Directors
TatsutoshiNakahata
JunyaToguchida
Executive Board
Research Departments
Department of
Reprogramming Science
Department of Cell
Clinical Application
Department of
Regulatory Science
Department Head
Department Head
Department Head
Department Head
ShinyaYamanaka
JunyaToguchida
TatsutoshiNakahata
ShinyaYamanaka
YasuhiroYamada
JunTakahashi
HaruhisaInoue
TakafumiKimura
YoshinoriYoshida
JunK.Yamashita
HidetoshiSakurai
TakashiAoi
MasatoNakagawa
KenjiOsafune
MegumuSaito
IsaoAsaka
KeisukeOkita
TomohisaKato
KazutoshiTakahashi
TaroToyoda
KnutWoltjen
TakuyaYamamoto
AkitsuHotta
AkiraWatanabe
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Department of Cell
Growth and
Differentiation
CiRA research building
Open laboratory
Faculty Council
Animal Research
Facility
Head
YasuhiroYamada
Research Strategy
Division
Administration
Division
Head
Head
HideyaHayashi
FusaoKoyama
Research
ManagementOffice
GeneralAffairs
Section
Contract
ManagementOffice
FinanceSection
IntellectualProperty
ManagementOffice
Facility for iPS Cell
Therapy (FiT) cell
processing center
InternationalPublic
Communications
Office
Head
TakafumiKimura
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Shinya Yamanaka
Profile
M.D., Ph.D.
Director, Department Head & Professor
Born in Osaka in 1962, Yamanaka received his
M.D. from the Kobe University School of
Medicine and completed the Ph. D. program at
the Osaka City University Graduate School,
Division of Medicine. He pioneered induced
pluripotent stem (iPS) cell research, reporting
the generation of these in mouse in 2006 and in
human in 2007.
Human iPS cells
Members
●
Michiko Nakamura
Professors
Shinya Yamanaka
Ayumi Ichikawa
Akira Ota ※
Ito Miyashita
Keiichi Nagai
●
※
Lecturers
Yoshinori Yoshida
●
Graduate Students
Kumiko Iwabuchi
Masatoshi Kajiwara
Masato Nakagawa
Akiko Fukuhara
Keisuke Okita
Takayuki Tanaka
Kazutoshi Takahashi
●
Researchers
Michiyo Koyanagi
Momoko Maekawa
Sarita Panula
Takahiro Sato
●
Technical Staff
Tomoko Ichisaka
Tomonori Nakamura
Hyenjong Hong
Koji Tanabe
Mari Onuki
Ren Shimamoto
Hidaka Yokota
Yuji Mochizuki
Masatoshi Nishizawa
Katsutaro Yasuda
Megumi Narita
Marie Muramatsu
Nanako Takizawa
Tatsuya Yamakawa
Aki Okada
Hayami Sugiyama
Midori Yokura
Hiroki Ikeda
Ran Shibukawa
Ikumi Kodanaka
Kazuyo Tamaoki
Masachika Iizuka
Sadamu Konishi
Yasuko Matsumura
Misato Nishikawa
●
Secretary
Rie Kato
Eri Nishikawa
Sayaka Takeshima
Yuko Otsu
Haruka Hasaba
Akiko Ohishi
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the method of generating these cells relied
on the delivery of four factors (Oct3/4,
Sox2, Klf4, and c-Myc) using retroviral
vectors, which led to concerns over the
possibility of tumorigenesis resulting from
the uncontrolled genomic integration of
these transgenes. Additionally, one of the
four original reprogramming factors,
c-Myc, is a known oncogene, which raised
further concerns of adverse effects.
Subsequent research has led to the
development of a plasmid-based method
for delivery of the reprogramming factors
that does not involve genomic integration,
as well as the finding that, in mice, the
safety of transplanted cells depends more
on the donor cell source than it does on the
reprogramming factors or generation
CiRA s goals over
the next decade
① Establishing fundamental
technologies and securing
intellectual property
② Using patient-derived iPS cells in
the development of drug discovery
techniques
③ Establishing an iPS cell bank for use
Mika Ohuchi
Yoshiko Sato
In 2006, we reported the first-ever
generation of induced pluripotent stem
(iPS) cells in mouse, and the following year
we were among the first to report the
generation of human iPS cells. In 2008, our
group developed a plasmid-based system
for the delivery of reprogramming factors
in the generation of mouse iPS cells. iPS
cells show great potential for applications
in drug discovery, and as a resource in
regenerative medicine, which has attracted
many labs from around the world to study
them. In order to realize this promise
however, much research still needs to be
done, such as developing methods for
optimized iPS cell production and safety
evaluation based on an understanding of
the mechanisms involved in
reprogramming differentiated cells to a
pluripotent state. Over the next decade,
CiRA will seek to: 1) establish fundamental
technologies and secure intellectual
property, 2) use patient-derived iPS cells in
the development of drug discovery
techniques, 3) establish an iPS cell bank for
use in regenerative medicine applications,
and 4) conduct preclinical and clinical
studies.
※ Specially appointed
Development of iPS cells
for clinical applications
In the early days of iPS cell research,
in regenerative medicine
④ Conducting preclinical and clinical
studies
CiRA ANNUAL REPORT 2010
methods used. Cells used in the clinical
setting must also be free of contaminants,
which had prompted study of appropriate
iPS cell culture methods as well. To date,
most methods for iPS cell derivation and
maintenance have relied on the use of
mouse feeder cells, but techniques have
now been developed for the generation of
autologous feeder cells from the same
cellular source as used for generating the
iPS cells themselves, which should be useful
in avoiding the problems associated with
xeno culture.
In 2010, we showed that it is possible to
use L-Myc instead of the problematic c-Myc
in generating iPS cells and that doing so
results in higher derivation efficiencies and
lower risks of tumor formation.
Research into technologies for the
generation of iPS cells is advancing at the
global level, and as a result there are many
varieties of iPS cells derived using different
combinations of source tissue,
reprogramming factors and delivery
methods. These methodological differences
have been shown to be linked to differences
in iPS cell properties and safety. In order to
identify the iPS cells best suited to drug
discovery and clinical applications and
develop global standards in the field, we are
conducting comparisons of iPS cells
generated using the various combinations
described above.
Study of the pathogenesis of
human diseases
and drug development
It is thought that by generating iPS cells
using a patient s somatic cells, it should be
possible to differentiate these into various
cell types and tissues affected by a given
disease. Particularly for cell types, such as
neurons and cardiac cells, which cannot be
obtained simply, this may represent a useful
resource for the study of disease etiology
and the development of new drugs, as well
as for toxicology studies.
At CiRA, we are working toward these
goals through the generation of patientderived iPS cells for in vitro disease
modeling and the study of disease origins
and mechanisms of progression, as well as
the development of therapeutic candidates
to control such diseases.
Establishment of iPS cell bank
for regenerative medicine
The use of autologous iPS cells should
make it possible to avoid rejection on
transplantation, but as the induction of
Global standard methods for generating and evaluating iPS cells
on the path to clinical development
The application of iPS cells in drug development and regenerative medicine will require
an understanding of reprogramming mechanisms, the development of safe derivation
methods, and systems to facilitate clinical use.
In 2010, the Yamanaka research group determined that Myc family factor L-Myc can
be substituted for the oncogene c-Myc, resulting in iPS cells with lower risks of tumor
formation, and a higher reprogramming efficiency.
They also developed a method for generating iPS cells without relying on mouse
feeder cells, using autologous fibroblasts. Detailed analysis of mouse iPS cells has
further revealed that the source tissue type plays an important role in determining iPS
cell safety.
In addition to conducting such comparisons to promote the development of global
standards for source cells and derivation methods, they are working on studies of
disease mechanisms and drug development using patient-derived iPS cells.
They are further working toward the establishment of a bank of human iPS cells
carrying HLA3 loci with lower risks of immune rejection for future clinical use.
pluripotency is a time-consuming process,
and newly derived cells need to be tested
for quality, they must be available in
advance if they are to be used in the rapid
treatment of acute medical conditions. The
generation of iPS cells safe for clinical use is
also expensive. All cells carry molecular
markers known as HLA (human leukocyte
antigen) signatures, which are analogous in
some ways to the ABO distinctions between
blood types. The HLA types work to
distinguish self tissue from non-self, which
is the basis for the rejection of transplanted
tissue by the immune system. There are,
however, rare homologies in HLA3 loci
that are linked with low rates of rejection.
By deriving iPS cells from persons
carrying these HLA types, it should be
possible to create a bank of iPS cells ready
for use in clinical applications. It has
been estimated that a set of 50 cell lines
representing these HLA3 versions would
provide approximately 80% coverage of
the Japanese population. Although it may
not be possible to obtain all 50 lines from
the onset, we will seek to develop a bank
of broad coverage iPS cells for clinical
use.
Publications
1. Nakagawa M, Takizawa N, Narita M, Ichisaka T, Yamanaka S.
Promotion of direct reprogramming by transformation-deficient Myc. Proc Natl
Acad Sci U S A. 107(32), 14152-14157, 2010.
2. Takahashi K, Narita M, Yokura M, Ichisaka T, Yamanaka S.
Human induced pluripotent stem cells on autologous feeders.
PLoS One 4, e8067, 2009.
3. Hong H, Takahashi K, Ichisaka T, Aoi T, Kanagawa O, Nakagawa M, Okita K,
Yamanaka, S.
Suppression of induced pluripotent stem cell generation by the p53-p21 pathway.
Nature 460, 1132-1135, 2009.
4. Miura K, Okada Y, Aoi T, Okada A, Takahashi K, Okita K, Nakagawa M, Koyanagi
M, Tanabe K, Ohnuki M, Ogawa D, Ikeda E, Okano H, Yamanaka S.
Variation in the safety of induced pluripotent stem cell lines.
Nat Biotechnol. 27, 743-745, 2009.
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Yasuhiro Yamada
Profile
M.D., Ph.D.
Head of Animal Research Facility & Professor
Born in Gifu City in 1972. Graduated from Gifu
University School of Medicine. Studied at the
Gifu University Graduate School of Medicine
(without completion of the course). Appointed
Assistant Professor at Gifu University School of
Medicine. Acquired PhD in Medicine and
Certified as a Pathologist. After working as a
Post-Doctoral Fellow at the Massachusetts
Institute for Biomedical Research, Massachusetts
Institute of Technology, and as a Lecturer and
then Associate Professor at Gifu University
Graduate School, assigned to the current
position in 2010. Since 2008, concurrently
assigned to PRESTO (Sakigake) Researcher of the
Japan Science and Technology Agency (JST).
Teratoma
Members
●
Professor
Yasuhiro Yamada
●
Assistant Professor
Akira Watanabe
●
Technical Staff
Tomoyo Ukai
Kiyoko Osugi
●
Graduate Students
Kotaro Onishi
Yuko Arioka
Kyoichi Hashimoto
●
Researcher
Yutaka Matsuda
●
Epigenetics Working Group
Miyuki Suzuki
Seiko Nishimoto
●
Secretary
Nao Nishimoto
Understanding the mechanism for cellular
reprogramming seems to be very useful in
preparing high quality iPS cells (induced
pluripotent stem cells) aimed at clinical
application.
It has been demonstrated that the
epigenetic regulation mechanisms,
involving DNA methylation, histone
modification, etc. and independent of DNA
base sequence, play important role in
regulation of gene expression. Cellular
reprogramming has been shown to be
accompanied by dynamic changes in
epigenetic modifications, indicating that
epigenetic regulation is important in
establishment of iPS cells. To date, however,
the exact mechanism for such regulations
remains to be clarified. The Yamada
Laboratory has set a goal of elucidating the
mechanisms for cellular reprogramming
through understanding of the epigenetic
regulation mechanisms.
Creation of an efficient and stable
iPS cell establishing system
Establishment of iPS cells requires time
on the order of weeks. Even after
introduction of reprogramming factors
into cells, reprogramming fails in most of
these cells. It is therefore desirable to
develop a more efficient iPS cell
establishing system to enable analysis of
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intermediate steps prior to establishment of
iPS cells. Furthermore, it is anticipated that
detailed analysis of iPS cell establishing
processes may be hampered by random
integration of the viral transgenes, the
number of transgene copies and changes in
expression level of each transgenes during
the course of iPS cell establishment. During
the current year, our laboratory has begun,
in collaboration with the Woltjen
Laboratory, development of a secondary
iPS cell establishing system making use of
the doxycycline-based gene expression
regulation system, with a goal of creating a
more efficient and stable iPS cell
establishing system. From now, we plan to
use these systems in examining how the
epigenetics-associated factors are involved
in cellular reprogramming.
Establishment of technology for
identification of epigenomic
modifications
It is now essential to identify
modificatory changes in epigenome during
the course of iPS cell establishment in a
concrete manner. The Epigenetics Working
Group led by Assistant Professor Akira
Watanabe is now attempting to establish
the technology for thorough identification
of epigenetic modifications. At present,
research is under way to determine the
Attempting to elucidate the mechanisms for cellular reprogramming
and to create an efficient iPS cell establishing system
Chimeric Mice
genomic sequences of methylated region
with a next-generation sequencer using the
methylated DNA fragments harvested with
MBD (methyl-CpG-binding domain)
protein. This technique has enables
genome-wide identification of the regions
of DNA methylation different between
somatic cells and iPS cells. From now, we
plan to identify epigenetic changes during
the course of iPS cell establishment
through analyzing changes in the binding
site of reprogramming factors (Oct3/4 and
so on) and in DNA methylation.
Thorough analysis of modificatory
changes in epigenome is expected to be
useful also in quality control of iPS cells.
Clarification of epigenetic
modifications in cancer through
establishment of cancer-derived
iPSC-like cells
The iPS cell establishing technology is
promising as a tool for altering the status of
At the Yamada Laboratory, efforts are being made to create a system enabling efficient
establishment of iPS cells through elucidating the mechanisms for regulation of
alterations in epigenetic modifications involved in cellular reprogramming. At present,
the laboratory is engaged in development of a secondary iPS cell establishing system
and in identification of changes in epigenome during the course of iPS cell
establishment with the use of such a system. In addition, development of epigenetic
analysis technology (particularly pertaining to methylated DNA sequences and histone
modifications) using a next-generation sequencer is now under way.
Furthermore, they have created cells similar to iPS cells by reprogramming the
colorectal tumor cells of a mouse model of familial adenomatous polyposis and
confirmed the changes of tumor cell-specific abnormal DNA methylation. This finding
suggests that even in cancer cells, genomic modification by methylation can be altered
markedly, providing a vital clue to development of a new approach of cancer treatment
through alteration of epigenetic modifications. They plan to attempt reprogramming of
other types of cancer and conduct further research into the significance of epigenetic
modifications in cancer cells.
epigenetic regulation. Many years ago
cancer cells were found to involve
abnormal gene sequences and abnormal
epigenetic modifications as represented by
abnormal DNA methylation. Recent studies
revealed that DNA methylation plays an
important functional role in carcinogenesis.
However, there are still many questions
pertaining to the importance of epigenetic
modifications in cancer. The Yamada
Laboratory has been attempting to
elucidate the significance and origin of
abnormal epigenetic modifications in
cancer cells by applying the iPS cell
establishing technology to cancer cells as a
tool for inducing alteration of epigenetic
regulation in cancer cells. During the
current year, we have attempted
reprogramming of colorectal tumor cells of
Apc Min mice, a mouse model of familial
adenomatous polyposis, and have
established iPSC-like cells which are
morphologically resemble pluripotent
stem cells. Analysis of DNA methylation
of tumor-derived iPSC-like cells revealed
that tumor-specific abnormal DNA
methylation was altered and that such
altered DNA methylation was further
changed by induction of differentiation
in tumor-derived iPSC-like cells. These
findings indicate that modifications of
DNA methylation can be altered
markedly even in tumor cells possessing
abnormal gene sequences, suggesting the
possibility of cancer treatment with a
target set at epigenetic modifications. In
addition, it has been becoming
increasingly evident that complete
reprogramming of tumor cells is difficult.
We plan to attempt tumor cell
reprogramming, expanding the scope of
coverage to other types of cancer.
HDF
ESC1
ESC2
ESC3
iPSC1
iPSC2
iPSC3
iPSC4
An example of DNA methylation analysis with a next-generation sequencer
Publications
1. Yamada Y, Aoki H, Kunisada T,
Hara A.
Rest promotes the early
differentiation of mouse ESCs but is
not required for their maintenance.
Cell Stem Cell 6(1):10-15, 2010.
2. H. Tomita, A. Hirata, Y. Yamada,
K. Hata, T. Oyama, H. Mori,
S. Yamashita, T. Ushijima, A. Hara.
Suppressive effect of global DNA
hypomethylation on gastric
carcinogenesis.
Carcinogenesis 31(9):1627-1633, 2010.
11
Yoshinori Yoshida
Profile
M.D., Ph.D.
Lecturer
Born in Kyoto Prefecture in 1973. Graduated
from Kyoto University School of Medicine.
Completed Kyoto University Graduate School of
Medicine. Acquired PhD in medicine. After
receiving training on cardiac catheterization and
therapeutic catheter intervention as a
cardiologist, began in 2002 to be engaged in
research on heart development and
regeneration at Kyoto University Graduate
School. Since 2007, conducting research on
induced pluripotent stem cells (iPS cells) at the
Institute for Frontier Medical Sciences and the
Center for iPS Cell Research and Application. The
goal of research is realization of myocardial
regenerative therapy with the use of iPS cells.
Measurement of field potential of cardiomyocytes
derived from human iPS cells
Members
●
Lecturer
Yoshinori Yoshida
●
Graduate Students
Hidaka Yokota
Masatoshi Nishizawa
Embryonic stem cells (ES cells), established
from human or mouse embryos, are
capable of proliferating while retaining
pluripotency. It has been shown that
introduction of reprogramming factors
(c-Myc, Oct3/4, SOX2, Klf4) into somatic
cells can induce iPS cells, which are
pluripotent like ES cells. iPS cells are
expected to be applicable to regenerative
medicine.
iPS cells can be established from
various types of somatic cells, and diverse
methods for their establishment have been
reported. However, iPS cells are not
identical in properties to ES cells, and the
properties of these iPS cells are considered
to vary among different cell lines.
Analysis of differences among cell
lines in the orientation of iPS cell
differentiation along a specific
direction (differentiation into
cardiomyocytes and blood cells)
We have been comparing and analyzing
the profiles of ES/iPS cells in induced
differentiation into mesoderm cells among
different cell lines, to elucidate the
mechanism determining the profiles of
pluripotent stem cells and to establish the
technique for generation and maintenance
of iPS cells optimal for clinical application
(use in regenerative medicine, etc.). It is
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known that treatment with cytokines at the
time of induction of ES/iPS cell
differentiation enables efficient
differentiation into cardiomyocytes. When
we attempted induction of differentiation
along a specific direction (differentiation
into cardiomyocytes) from ES/iPS cells, the
efficiency of cardiomyocyte induction
varied greatly among different cell lines. At
present, we are conducting research into
the mechanisms for such a difference in the
orientation of differentiation among cell
lines.
At the same time, we have also
confirmed that in induction of directional
differentiation into blood cells (cells of the
lateral plate mesoderm like
cardiomyocytes), the orientation of
differentiation differs among cell lines. We
are studying the mechanisms for such a
difference as well.
Exploration of cell culture settings
and factors enabling more efficient
reprogramming
The efficiency of reprogramming from
somatic cells to pluripotent stem cells is
determined by various factors. We found
that cell culture under hypoxic condition
(oxygen concentration: 5%) improved the
efficiency of human and mouse iPS cell
establishment. It was shown that
Derivation of cardiomyocytes from human iPS cells by directed differentiation (Left:phase contrast, middle:nuclei(DAPI), right:Troponin T)
reprogramming in low oxygen culture
enables induction of iPS cells from mouse
embryonic fibroblasts with the use of two
factors (Oct3/4 and Klf4), and that the
expression of genes specific to pluripotent
stem cells (Oct3/4, Nanog, etc.) was
increased in the cells reprogrammed in low
oxygen culture. Although the exact
mechanism for improvement in the
efficiency of reprogramming in low oxygen
culture remains unclarified, alterations in
the expression of these genes may be
involved.
At present, we are also analyzing the
impact of various intracellular signaling
pathways on the reprogramming efficiency.
Attempts of clinical application of
iPS cell research findings
1. Research on disease-specific iPS cells
Jointly with the Department of
Cardiology, Kyoto University, we have been
conducting research on disease-specific iPS
cells established from somatic cells
collected from patients with hereditary
arrhythmias and myocardial diseases. To
date, fibroblasts have been collected from a
total of 37 patients (20 with hereditary
arrhythmias and 20 with myocardial
diseases), and iPS cells have been
established from these cells one after
another. At present, we are conducting
functional analysis of myocardial cells
induced from these iPS cells.
2. Realization of myocardial regenerative
therapy with iPS cell-derived myocardial
cells
We consider that cell transplantation
from the endocardial side of heart with
the use of a catheter is an important
approach to cell transplant therapy, in
addition to the approach from the
pericardial side of heart by means of
open heart surgery, because this
approach is less invasive and can be
repeated. Jointly with the Department of
Cardiology, Kyoto University, we have
started research for creation of a model
of catheterized cell transplantation with
the use of medium-size animals.
Realization of myocardial regenerative therapy through elucidation
of the mechanism for differentiation from pluripotent stem cells
and improvement in the efficiency of somatic cell reprogramming
Publications
Lecturer Yoshinori Yoshida, previously specializing in cardiology, is now conducting
research towards the goal of establishing regenerative medicine with the use of
myocardial cells. During the current fiscal year, he attempted to elucidate the
mechanism for differentiation of ES cells and iPS cells into diverse cells and
demonstrated that the efficiency of induction of differentiation differed among cell
lines.
He additionally found that the reprogramming of cells and the expression of genes
in reprogrammed cells were more efficient in oxygen poor settings. From now, he plans
to analyze the intracellular signal transduction pathways determining the efficiency of
reprogramming.
Research is also under way on establishing iPS cells from somatic cells of patients
with hereditary arrhythmias and myocardial diseases and inducing their differentiation
into myocardial cells. A study of animal models to develop less invasive cell transplant
therapy with the use of a catheter has also started.
1. Yoshida Y, Yamanaka S.
iPS cells: A source of cardiac
regeneration.
J Mol Cell Cardiol. 50(2):327-332, 2010.
2. Yoshida Y, Yamanaka S.
Recent stem cell advances: induced
pluripotent stem cells for disease
modeling and stem cell-based
regeneration.
Circulation 122(1):80-87, 2010.
3. Yoshida Y, Takahashi K, Okita K,
Ichisaka T, Yamanaka S.
Hypoxia enhances the generation of
induced pluripotent stem cells.
Cell Stem Cell 5(3):237-241, 2009.
13
Masato Nakagawa
Profile
Ph.D.
Lecturer
Born in Ashiya City, Hyogo Prefecture in 1975.
Graduation from Department of Chemistry,
Faculty of Science and Technology, Sophia
University in 1997. Completed Nara Institute of
Science and Technology Graduate School of
Biological Sciences in 2002. Acquired PhD in
Bioscience. As a graduate student, engaged in
research on cell-cell adhesion and the signal
transduction systems involved, demonstrating
hemophilic binding of E-cadherin (a cell
adhesion molecule) and activation of Rac1 small
GTPase serving as intracellular molecular
switches. Thereafter, began to elucidate the
molecular mechanism of pluripotency for ES
cells. Currently engaged on methods enabling
safe and efficient establishment of iPS cells.
A mouse with germline transmission by mouse
L-Myc iPS cells
Members
●
Lecturer
Masato Nakagawa
●
Graduate Students
Tomonori Nakamura
Hayami Sugiyama
Application of pluripotent stem
cells to regenerative medicine
Human ES cells have the potential of
differentiating into every type of cells
constituting the human body
(pluripotency) and have thus been viewed
as a resource applicable to regenerative
medicine. However, clinical application of
ES cells involves problems related to
practical procedure on the use of fertilized
eggs, etc. and possible host rejection to cell
transplantation. One possible means of
resolving these issues is to create
pluripotent cells directly from somatic cells.
We have succeeded in establishing iPS cells
by introducing 4 transcription factors
(Sox2, Oct3/4, Klf4 and c-Myc) with
retrovirus into somatic cells. The thus
established iPS cells have been shown to
have properties quite similar to ES cells.
Tumorigenic risk of iPS cells and
involvement of c-Myc
Tumorigenesis was frequently seen in
chimeric mice derived from mouse iPS
cells. Detailed analysis of these mice
demonstrated that tumorigenesis was
attributable to reactivation of retroviral
c-Myc inserted into the host genome.
Clinical application of iPS cells in this form
was thus considered to involve high risk.
For this reason, we prepared a protocol for
14
iPS cell establishment without involving the
retrovirus carrying c-Myc. With this
protocol, we have succeeded in establishing
Myc minus iPS cells from human and
mouse somatic cells. Tumorigenesis was
almost absent in chimeric mice derived
from Myc minus iPS cells, but recent
studies suggested that the quality of Myc
minus iPS cells is lower than c-Myc iPS cells.
Establishment of high safety iPS
cells with L-Myc
The risk for tumorigenesis is a
significant open issue in clinical application
of iPS cells. We therefore explored factors
which could serve as substitutes for c-Myc
in establishing iPS cells.
First, functional analysis of Myc gene
was conducted. There are several genes of
the Myc family. Both mouse and human
have c-Myc, N-Myc and L-Myc. Major
functional domains are common among
family genes, but differences are seen in the
other regions among these genes. With this
in mind, we analyzed the function of these
Myc family genes in establishment of iPS
cells to examine whether or not genes other
than c-Myc could serve as substitute factors.
This analysis revealed that the use of L-Myc
(identified among the Myc family genes)
enables more efficient establishment of
mouse iPS cells than the use of c-Myc.
Exploration of genes as
substitutes for oncogenes
used in iPS cell establishment
to suppress tumorigenesis
Human iPS cells established with L-Myc
When chimeric mice were generated with
L-Myc iPS cells and followed for long
periods of time, like those generated with
c-Myc, tumorigenesis was seldom seen. It
was thus shown that L-Myc can induce iPS
cells with high safety. The number of iPS
cell colonies appearing during the course of
human iPS cell establishment and its
efficiency were also higher with L-Myc than
with c-Myc.
These results indicate that the use of
L-Myc enables efficient establishment of iPS
cells with high safety. The iPS cells
established with L-Myc are promising as
cells for clinical application.
Perspectives for research on iPS
cells in the future
One effective way of utilizing iPS cells
is to use them as patient-derived diseasespecific cells. This pertains to establishment
of iPS cells from the skin fibroblasts
collected from patients with hereditary
brain/nerve disease. If differentiation of
nerve cells from these iPS cells is induced,
we may obtain a model of cells carrying the
patient-derived phenotype caused by their
genetic anomalies. The use of this model
may enable studies which have been
difficult conventionally (e.g., evaluation of
drug efficacy, and toxicological assessment).
In addition, if used for large-scale
screening, this model may serve as a very
useful resource.
Much is now expected of applying iPS
cells to regenerative medicine, and
competition over research on iPS cells in
Japan and overseas is intensifying.
However, the mechanism for iPS cell
establishment has not yet been clarified
well, and research providing insights into
such basic aspects will be increasing in
importance on. In particular, it seems
essential to have sufficient understanding
of the safety of iPS cells and to resolve
safety problems.
Lecturer Masato Nakagawa has been
laying emphasis on research designed
to elucidate the mechanism for iPS cell
establishment, particularly on
verification of iPS cell safety and
resolving safety problems.
Because iPS cell establishment
employs c-Myc (an oncogene) as one of
the four transcription factors, there
have been concerns over tumorigenesis
since the start of this kind of research.
In practice, tumorigenesis frequently
occurs in the chimeric mice generated
with mouse iPS cells because of
reactivation of retroviral c-Myc inserted
into the host genome. To resolve this
issue, Lecturer Nakagawa and his
coworkers explored factors which could
serve as substitutes for c-Myc. They
found that among the Myc family
genes, L-Myc enables more efficient
establishment of iPS cells, seldom
resulting in tumorigenesis in the
chimeric mice generated with L-Myc iPS
cells. This finding has advanced iPS cell
research to a stage closer to clinical
application.
iPS cells still have a lot of hidden
secrets. He believes that basic research
can unlock them, which hopes to
contribute to the development of
clinical applications.
Publications
1. Nakagawa M, Takizawa N, Narita M, Ichisaka T, Yamanaka S.
Promotion of direct reprogramming by transformation-deficient Myc.
Proc Natl Acad Sci U.S.A. 107(32), 14152-14157, 2010.
M, Tanabe K, Ohnuki M, Ogawa D, Ikeda E, Okano H, Yamanaka S.
Nat Biotechnol. 27:743-745, 2009.
Yamanaka S.
Nature 460, 1132-1135, 2009.
4. Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka S.
Generation of mouse induced pluripotent stem cells without viral vectors.
Science 322:949-953, 2008.
5. Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K,
Mochiduki Y, Takizawa N, Yamanaka S.
Generation of induced pluripotent stem cells without Myc from mouse and
human fibroblasts.
Nat Biotechnol. 26:101-106, 2008.
15
Keisuke Okita
Profile
Ph.D.
Born in Gose City, Nara Prefecture in 1975.
Graduated from Graduate School of Veterinary
Medicine in Hokkaido University. Completed the
doctor course of Graduate School of Medicine in
Kumamoto University. Appointed JST (Japan
Science and Technology Agency) researcher, JSPS
(Japan Society for the Promotion of Science)
research fellow, and assistant professor in
Center for iPS Cell Research and Application of
iCeMS (Institute for Integrated Cell-Material
Sciences) in Kyoto University, and lecturer in
iCeMS before taking to the current position.
Lecturer
Chimera Mouse
Members
●
Lecturer
Keisuke Okita
●
Graduate Students
Kumiko Iwabuchi
Hong Hyenjong
Ren Shimamoto
Tatsuya Yamakawa
Development of iPS cell induction
methods without genomic
modification
The technology for creation of iPS cells
is still developing. For example, c-Myc, one
of the reprogramming genes, is also known
as an oncogene, and we have demonstrated
Human iPS cells
16
that exogenous c-Myc inserted into the
genome of iPS cells by retrovirus vector
increased the risk of tumorigenesis. To
resolve this problem, we have been
developing method without using c-Myc
and method for creation of mouse iPS cells
without genomic modification. In 2010, we
further advanced these techniques towards
the goal of development of safe method for
human iPS cell establishment. Through the
evaluation of several combinations of
reprogramming genes and various methods
for their transfer, we have established iPS
cells from multiple cell lines. Because high
reproducibility is a very important
requirement for clinical application, we are
now attempting to establish an easy
protocol which will allow investigators to
establish iPS cells without difficulty. Recent
studies have been reported iPS cell
induction by Sendai virus vector, and direct
transfer of RNA or protein. We plan to
conduct follow-up studies on these
methods. We will also evaluate the
differences among these iPS cells.
Establishment of iPS cells from less
invasive tissues
Fibroblasts are often used for
establishment of iPS cells. To induce
patient-specific iPS cells and to make iPS
cell bank, it seems desirable to establish a
method for iPS cell creation from various
tissues which can be collected in a less
invasive manner. In 2010, we reported that
human iPS cells can be prepared from
gingival cells in collaboration with Drs.
Hiroshi Egusa and Hirofumi Yatani in
Osaka University. This finding suggests the
possibility of iPS cell induction from
conventionally discarded tissues after
dental treatment (e.g., treatment of
periodontal disease and implant therapy).
In addition, several reports have
demonstrated that iPS cells could be
established from pulp stem cells of wisdom
teeth, mesenchymal stem cells of
subcutaneous fat tissue, keratinocytes,
umbilical cord blood and peripheral blood.
We plan to confirm these reports with a
goal of developing practical methods of iPS
establishment.
Epigenetic analysis
Recent evidences have unveiled that
mouse and human iPS cells remain some
epigenetic status of their original tissue. For
example, iPS cells derived from blood cells
can easily re-differentiate into blood cells,
while iPS cells derived from nerve cells
differentiate less efficiently. Considering
that the treatment with a DNA methylation
inhibitor (5-azacytidine) and a histone
deacetylation inhibitor (trichostatin A)
improved the efficiency of the
differentiation potential of neuron-derived
iPS cells, it is likely that DNA methylation
Ongoing efforts to develop a safe, efficient and less invasive
method of iPS cell establishment
At the Okita Laboratory, research is now under way on methods for establishment of
iPS cells without oncogene insertion into the genome. In the current year, the group
explored and evaluated various combinations of reprogramming genes and methods
for their introduction. The lab. are also attempting to establish iPS cells from less
invasive cells and have succeeded in establishment of iPS cells from gingival cells with
Dr. H. Egusa in Osaka University.
iPS cells remain, at least, in part of epigenetic status of their origin, and modulation
of DNA methylation and histone acetylation improved the differentiation potential of
iPS cells. Last year, they reported the methylation profile of mouse iPS cells with Dr. K.
Shiota in University of Tokyo.
Analysis of intracellular signals which determine the reprogramming process and
gene expression during iPS cell induction are also under way. They would like to
identify the reasons which are responsible for inter-clone differences of iPS cells and to
determine the safest and efficient way for iPS cell establishment.
and histone acetylation are involved in the
memory of their source cells. Thus we
focus on DNA methylation, and have
reported the DNA methylation profile of
mouse iPS cell with Dr. Kunio Shiota in
University of Tokyo. On the basis of DNA
methylation patterns, we are now studying
how they affect the properties of iPS cells.
elucidate the reprogramming process
through analysis of gene expression
profile after the introduction of
reprogramming genes. These studies have
been gradually revealing processes of iPS
cell induction. We are interested in the
differences among multiple iPS clones
and method which would enable us to
establish iPS cells with highest safety.
Elucidation of the mechanism
underlying reprogramming
Last year, we demonstrated that the
suppression of p53-p21 pathway increased
the efficiency of iPS cell establishment. We
are now analyzing downstream factors of
the signal. We are also attempting to
Publications
1. Egusa H, Okita K, Kayashima H, Yu G, Fukuyasu S, Saeki M, Matsumoto T,
Yamanaka S, Yatani H.
Gingival fibroblasts as a promising source of induced pluripotent stem cells.
PLoS One 5(9):e12743, 2010.
2. Sato S, Yagi S, Arai Y, Hirabayashi K, Hattori N, Iwatani M, Okita K, Ohgane J,
Tanaka S, Wakayama T, Yamanaka S, Shiota K.
Genome-wide DNA methylation profile of tissue-dependent and differentially
methylated regions (T-DMRs) residing in mouse pluripotent stem cells.
Genes Cells (6):607-618, 2010.
3. Okita K, Hong H, Takahashi K, Yamanaka S.
Generation of mouse-induced pluripotent stem cells with plasmid vectors.
Nat Protoc. 5(3):418-428, 2010.
4. Yoshida Y, Takahashi K, Okita K, Ichisaka T, Yamanaka S.
Hypoxia enhances the generation of induced pluripotent stem cells.
Cell Stem Cell 5(3):237-241, 2009.
Yamanaka S.
Nature 460, 1132-1135, 2009.
17
Kazutoshi Takahashi
Profile
Ph.D.
Lecturer
Born in Hiroshima City in 1977. Graduated from
Doshisha University Faculty of Engineering.
Completed the Nara Institute Of Technology and
Science Graduate School of Biological Sciences.
Acquired PhD in Bioscience (2005). Appointed
JSPS (Japan Society for the Promotion of Science)
Research Fellow and Teaching Assistant at the
Institute for Frontier Medical Sciences and the
Center for iPS Cell Research and Application
before assigned in April 2010 to the current
position.
Human ES cells having differentiated into nerves in
suspension culture
Members
●
Lecturer
Kazutoshi Takahashi
●
Graduate Students
Koji Tanabe
Takayuki Tanaka
Mari Ohnuki
Marie Muramatsu
18
This group has been conducting research
with a goal set at standardization of iPS
cells and elucidation of the molecular
mechanism for self-renewal of pluripotent
stem cells. During the current year, the
following studies have been carried out.
1. Analysis of iPS cells showing
resistance to differentiation
Ensuring the safety is an issue of first
priority before clinical application of
human iPS cells in the future. Studies using
mouse iPS cells have shown that some
clones resist the induction of differentiation
and that the residual undifferentiated cells
are responsible for tumorigenesis after
transplantation of iPS cells. With this
finding in mind, we have been analyzing
the resistance of human iPS cells to
differentiation.
When multiple ES cell lines and more
than 20 human iPS cell lines were
compared by means of microarray analysis,
there was no significant difference among
these cell lines. Following this result, we
newly created a system for quantification of
residual undifferentiated cells after
induction of differentiation and evaluated
each clone with this system. This evaluation
revealed that the clones can be roughly
divided into two groups (a group of clones
relatively rich in residual undifferentiated
cells and a group of clones with a small
number of residual undifferentiated cells
comparable to that in ES cells. We then
induced differentiation of each clone into
nerve cells to examine whether or not these
results would correlate with the safety of
iPS cells. After 14-day induction of
differentiation, most cells of each clone
differentiated into PSA-NCAM positive
nerve cells. For most of the cell lines,
including ES cells, the percentage of cells
positive as to Oct3/4 (an indicator of
undifferentiated cells) was only about 1%.
However, of all iPS cell lines tested, 4 clones
were found to have a 10% or higher
percentage of Oct3/4 positive residual
undifferentiated cells in a well reproducible
manner.
From now, we will attempt to elucidate
the molecular mechanism through detailed
analysis of these cell lines exhibiting
resistance to differentiation.
2. Establishment of iPS cells with a
goal of clinical application
During the current year, we have made
two attempts of developing methods for
establishment and maintenance of iPS cells,
bearing in mind their clinical application in
the near future. The first attempt pertained
to the use of mouse-derived feeder cells
which had conventionally used for
establishment of human iPS cells. From the
viewpoint of clinical application, it is
desirable to eliminate unreliable
components derived from animals as far as
possible. For this reason, we attempted to
utilize human skin-derived fibroblasts not
only as a source of iPS cells but also as
feeder cells for establishment and
maintenance of iPS cells. In this attempt, it
was possible to establish and maintain iPS
cells on the self-feeder with all of the 4
skin-derived fibroblast lines tested.
However, with 3 of the fibroblast lines, it
was not possible to maintain the
undifferentiated status of ES cells serving as
feeder cells. These results suggest that
utilization of self-feeder cells as a means of
reducing the animal-derived components is
valid.
The second attempt pertained to
creation of dental pulp stem cell-derived
iPS cells jointly with Gifu University.
Dental pulp stem cells are advantageous in
that they can be established relatively
noninvasively from the tissues of wisdom
tooth, etc. which have conventionally
discarded without utilization. We
established iPS cells from 6 dental pulp
stem cell lines of different origins and
found that the efficiency for establishment
of iPS cells tended to be higher with cell
lines at lower stages of differentiation than
with skin fibroblasts. The iPS cells derived
from these dental pulp stem cells exhibited
gene expression patterns and pluripotency
not inferior to those of ES cells and skin
fibroblast-derived iPS cells. These results
indicate that dental pulp stem cells are one
Attempt of standardization of iPS cells through elucidation
of the molecular mechanism for induction of differentiation from
pluripotent stem cells
It has been shown that transplantation of undifferentiated mouse iPS cells can lead to
carcinogenesis. Thus, inducing differentiation in a reliable manner is needed so that iPS
cells can be applied clinically. Bearing this in mind, the Takahashi Laboratory has
developed a system for counting undifferentiated cells remaining after induction of
differentiation and analyzed with this system the differences among multiple ES and iPS
cell lines. In this way, 4 clones of iPS cell lines having a large number of residual
undifferentiated cells have been identified. From now, they will elucidate the molecular
mechanism for resistance of these clones to differentiation.
They have additionally reported the possibility of reducing the animal-derived
components by using human skin-derived fibroblasts (having the potential of becoming
human iPS cells) instead of the conventional mouse-derived cells as the feeder cells
(employed as the base for culture during establishment and maintenance of human iPS
cells). In a research jointly conducted with Gifu University, they have succeeded in
establishing iPS cells with high efficiency and quality derived from dental pulp stem cells.
Furthermore, they have obtained experimental results suggesting that introduction
of gene LIN28 elevates the reprogramming efficiency through its interactions with
micro-RNA. We are now conducting further analyses on this finding.
of the promising candidates for cell bank
sources in the future.
3. Functional analysis of factors
capable of elevating the efficiency
of reprogramming
This group has been identifying
multiple factors which can increase the
efficiency of reprogramming. During the
current year, the roles of LIN28 (one of
such factors) during reprogramming were
studied in detail. Introduction of LIN28
together with Oct3/4, Sox2, Klf4 and c-Myc
into human fibroblasts resulted in
improved efficacy of iPS cell colony
formation. However, with 5 of the 10
fibroblast lines, the number of iPS cell
colonies formed did not increase. A mutant
LIN28 (lacking the cold-shock domain in
the protein) exerted efficacy comparable
to that of the wild type LIN28, while
another mutant lacking the zinc finger
domain failed to exert efficacy. These
results suggest that interaction with
micro-RNA is an important activity of
LIN28 during the course of
reprogramming. In addition, it was
shown that the size of the iPS cell colony
formed in the presence of LIN28
expression was significantly larger than
that in the control group. There are
various views which can possibly explain
this phenomenon, and we are currently
conducting analyses to explain this
phenomenon definitely.
Publications
Picture of immunostained human iPS cells. Oct3/4 (an indicator of undifferentiated cells) is red. BrdU (an
indicator of cell proliferation) is green.
1. Tamaoki N, Takahashi K, Tanaka T,
Ichisaka T, Aoki H, TakedaKawaguchi T, Iida K., Kunisada T,
Shibata T, Yamanaka S, and Tezuka
K. Dental Pulp Cells for Induced
Pluripotent Stem Cell Banking.
J Dent Res. 89(8):773-778, 2010.
2. Takahashi K.
Direct reprogramming 101.
Dev Growth & Differ. 52(3): 319-333,
2010.
3. Takahashi K, Narita M, Yokura M,
Ichisaka T, Yamanaka S.
Human induced pluripotent stem
cells on autologous feeders.
PLoS ONE 4(12): e8067, 2009.
19
Knut Woltjen
Profile
Ph.D.
Birthdate: June 10, 1976
Birthplace: Edmonton, Alberta, Canada
1994-1998, BSc Honours, Molecular Genetics,
University of Alberta, Edmonton, Alberta,
Canada
1998-2006, Ph.D., Biochemistry and Molecular
Biology, University of Calgary, Alberta, Canada
2001-2003 Research Exchange, Kyushu
University, Fukuoka, Japan
2006-2009, Post Doctoral Fellow, Medical
Genetics, Samuel Lunenfeld Research Institute,
Toronto, Ontario, Canada
2009 – 2010, Manager, Ontario Human iPS Cell
Facility, The Hospital for Sick Children Research
Institute, Toronto, Ontario, Canada
2010 – present, Assistant Professor, Principal
Investigator, Center for iPS Cell Research and
Application (CiRA), Institute for Integrated
Cellular and Materials Sciences (iCeMS), Kyoto
University, Japan
Assistant Professor
Members
●
Assistant Professor
Knut Woltjen
●
Technical Staff
Ryoko Hirohata
Rumi Mochida
●
Secretary
Overview
Induced pluripotent stem cell (iPSC)
research is predicted to have a profound
impact on medical research and
regenerative medicine. Prior to clinical
applications in cellular therapy, access to
patient-specific, differentiated iPSC
derivatives is revolutionizing our regard for
in vitro cell-based human disease modeling
and drug screening. The Woltjen Lab is
focused on developing technologies and
research platforms for interrogating the
mechanism of somatic cell reprogramming
and plasticity, as well as improving human
disease models through augmented iPSC
derivation and genome engineering.
Erika Moriguchi
Human iPS cells expressing the mCherry fluorescent reporter from a piggyBac transposable element.
20
A
patients
B
C
D
E
F
G
H
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e12
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KCNQ1 exons
WAVE genotyping data revealing a common SNP (single nucleotide polymorphism) in exon 13 of Patient D s potassium channel gene, KCNQ1.
Developing Analytical
Reprogramming Tools
My prior research used piggyBac (PB)
transposons as novel, non-viral
reprogramming vectors (Woltjen et al.
Nature 2009; Kaji et al., Nature 2009). Here,
we employed drug-inducible transgene
regulation, allowing the production of iPSC
lines that may be cyclically differentiated
and (re)reprogrammed. Using this model
system, and in collaboration with CiRA
researchers (Dr. Yamada, Dr. Yamamoto),
we are revealing changes in transcriptional
networks and cell-cell interactions that
occur during the reprogramming process;
changes which may be applied to improve
upon current human reprogramming
standards.
Developing Methods for Precise
Genomic Modification
Homologous recombination allows
specific modification of human or mouse
stem cell genomes to elicit functional
changes. Such modifications can be
designed to disrupt normal genes or correct
mutant genes. Beyond its role as a factor
delivery vector in reprogramming, PB
transposition is a unique method of
transgenesis, loaning to its ability to remove
itself seamlessly from the genome.
Exploiting transposition and site-specific
DNA recombination tools, my lab at CiRA
is developing novel approaches to genetic
engineering that will enable more accurate
disease modeling and may eventually lead
to autologous cellular therapies.
Reprogramming and genetic engineering using a gene delivery
system that avoids permanent genomic modification
Using piggyBac transposons - mobile DNA sequences capable of jumping between sites
in the genome - as a delivery system for genetic factors, it is possible to avoid problems
associated with permanent integration of transgenes into the genome.
Knut Woltjen has developed a technique for reprogramming and differentiating cells
using the piggyBac system, achieving improved methods for generating modification-free
iPS cells and iPS cell derivatives. His lab is now investigating alternative methods for
genome engineering combining homologous recombination and transposon technology.
These methods may facilitate gene correction in patient-derived iPS cells, enabling the
validation of cellular models of human pathology, and even future clinical applications of
autologous induced pluripotent stem cells.
Publications
1. Monetti C, Nishino K, Woltjen K and
Nagy A.
PhiC31 integrase facilitates genetic
approaches combining multiple
recombinases.
Methods 2010. in press
2. Samavarchi-Tehrani P, Golipour A,
David L, Sung H-K, Beyer T A, Datti A,
Woltjen K, Nagy A, Wrana J L.
Functional Genomics Reveals a BMP
Driven Mesenchymal-to-Epithelial
Transition in the Initiation of Somatic
Cell Reprogramming.
Cell Stem Cell 7(1):64-77, 2010.
3. O'Malley J, Woltjen K, Kaji K.
New strategies to generate induced
pluripotent stem cells.
Curr Opin Biotechnol. 20(5):516-521,
2009.
4. Kaji K, Norrby K, Paca A, Mileikovsky
M, Mohseni P, Woltjen K.
Virus-free induction of pluripotency
and subsequent excision of
reprogramming factors.
Nature 458, 771-775, 2009.
5. Woltjen K, Michael I P, Mohseni P, Desai
R, Mileikovsky M, Hämäläinen R,
Cowling R, Wang W, Liu P,
Gertsenstein M, Nagy A.
piggyBac transposition reprograms
fibroblasts to induced pluripotent stem
cells.
Nature 458, 766-770, 2009.
21
Takuya Yamamoto
Profile
Ph.D.
Assistant Professor
Born in Osaka City in 1977. Graduated from
Faculty of Science, Kyoto University. Completed
the Graduate School of Biostudies, Kyoto
University. Acquired PhD in Life Science.
Engaged in research on MAP-kinase-related
signaling pathways and cell cycle using
Biochemistry and Bioinformatics at the
Laboratory of Signal Transduction (Eisuke
Nishida Laboratory), the Division of Integrative
Life Sciences of Graduate School of Biostudies,
Kyoto University. Assigned in April 2009 to the
current position.
Members
●
Assistant Professor
Takuya Yamamoto
●
Researcher
Masamitsu Sone
●
Technical Staff
Mio Kabata
Toshiko Sato
●
Graduate Students
Sho Ohta
Hiroki Ikeda
●
Secretary
Erika Moriguchi
Elucidation of the molecular mechanisms
underlying processes of iPS cell induction
is one of the important steps for
application of iPS cells to regenerative
medicine. Studies conducted to date
demonstrated that the phenomena
occurring in the nuclei (e.g., regulation of
transcription and epigenetic modification)
play key roles in these processes. In the
field of life science, recent remarkable
advances in analyzers as represented by
microarray and next-generation
sequencers have made it possible to obtain
huge volumes of data in short time. Our
group sets a primary goal at integrative
clarification of processes of the iPS cell
induction through analysis of the entire
genome with diverse approaches. During
the current year, we conducted
transcriptome analysis of iPS and ES cells
with a next-generation sequencer, and
developed a method for rapid
identification of retrovirus insertion sites
of various iPS cell lines.
1. Transcriptome analysis of
pluripotent stem cells with a nextgeneration sequencer
Alternative splicing, i.e., formation of
multiple transcription products from a
single gene, is seen with 95% or more
genes of mammals. At each stage or tissue
22
of development, alternative splicing is
regulated, often resulting in formation of
multiple proteins with varying activities
from a single gene. To date, numerous
studies involving transcriptome analysis
of pluripotent stem cells have been
carried out, mostly using microarray.
Because the microarray technology is
designed to measure the expression level
at only a portion of the entire region, it
was difficult to examine the expression
level of each splicing variant precisely.
The next-generation sequencer recently
introduced has overcome such a
shortcoming of microarray and is capable
of conducting transcriptome analysis to
yield an entire view of transcription.
Our group has created a full-length
cDNA library for pluripotent stem cells
(iPS cells and ES cells) and somatic cells
by the oligo-capping method and has
been conducting transcriptome analysis
with a next-generation sequencer SOLiD.
To date, we have devised a unique
algorithm capable of efficiently
identifying splicing variants and have
succeeded in identifying splicing variants
specific to pluripotent stem cells.
Furthermore, by examining cDNA of
each tissue, we have demonstrated that
about half of these splicing patterns in
pluripotent stem cells are similar to those
in testis. In addition, using the
bioinformatic technique, we have found
exon consensus sequences that might
characterize the splicing in pluripotent
stem cells.
2. Development of a method for
rapid identification of retrovirus
insertion sites of iPS cells with a
next-generation sequencer
Recently, reports on methods of iPS
cell induction without using retrovirus
vectors have been published one after
another. However, for the reason of
efficiency in iPS cell induction, methods
using retrovirus vectors are still used
frequently for induction of iPS cells.
Because the methods of iPS cell induction
with retrovirus vectors involve insertion of
a viral genome, the virus insertion sites
need to be taken into account when
research is conducted on
pathophysiological analysis or
development of new disease treatment
methods by using patient-derived iPS cells.
Our group has developed a technique
capable of precisely identifying the virus
insertion sites of iPS cell clones at a time.
Using this technique, we have identified
the virus insertion sites for multiple
patient-derived iPS cell lines and evaluates
these sites. The results from these studies
suggest that there is no virus insertion sites
specific to any particular disease, that the
virus is often inserted into the vicinity of
the transcription start sites or the first or
second intron, and that the level of gene
expression at the insertion sites (at least
the mRNA level) does not differ
significantly from that of ES cells.
Elucidation of processes of the iPS cell induction with the use of
next-generation sequencers
At the Yamamoto Laboratory, next-generation sequencers have been fully utilized for
transcriptome analysis of mRNA and transcription products of iPS and ES cells and rapid
identification of retrovirus insertion sites of iPS cells, with the goal of elucidating the iPS
cell induction processes.
Within the nucleus of cells, the necessary part of genetic information on DNA is cut
out and joined (splicing), resulting in a variety of mRNA (splicing variants). The group
have created full-length cDNA (induced DNA reflecting the full length of mRNA) for
each of iPS, ES and somatic cells and made them into a library for comparison. In this
way, the Yamamoto Lab. have identified splicing variants unique to iPS cells or ES cells.
Furthermore, they have developed a technique for identifying the viral genome
insertion sites following gene transfer on retrovirus vectors at the time of establishment
of iPS cells. Using this technique, they have identified sites where viral genome insertion
is likely to occur. They have additionally reported that the viral genome insertion sites
did not differ among different diseases when comparison was made among diseasespecific iPS cells, and that the mRNA expression level at the insertion sites did not differ
between iPS and ES cells.
Publications
1. Sunadome K, Yamamoto T, Ebisuya M, Kondoh K, Sehara-Fujisawa A, and Nishida E.
ERK5 Regulates Muscle Cell Fusion through Klf Transcription Factors.
Dev Cell. 20, 192-205, 2011.
2. Endo T, Kusakabe M, Sunadome K, Yamamoto T, and Nishida E.
The Kinase SGK1 in the Endoderm and Mesoderm Promotes Ectodermal Survival
by Down-Regulating Components of the Death-Inducing Signaling Complex.
Science Signal. 4, ra2, 2011.
3. Honjoh S, Yamamoto T, Uno M, and Nishida E.
Signalling through Rheb mediates intermittent fasting-induced longevity in C.
elegans.
Nature 457, 726-730, 2009.
4. Ebisuya M, Yamamoto T, Nakajima M, and Nishida E.
Ripples from neighbouring transcription.
Nature Cell Biol. 10, 1106-1113, 2008.
5. Yamamoto T, Ebisuya M, Ashida F, Okamoto K, Yonehara S, and Nishida E.
Continuous ERK activation downregulates antiproliferative genes throughout G1
phase to allow cell-cycle progression.
Curr Biol. 16, 1171-1182, 2006.
MEF
iPS
ES
Identification of splicing variants specific to iPS cells and ES cells.
23
Akitsu Hotta
Profile
Ph.D.
Born in Nagoya City in 1978. Graduated from
Department of Engineering, Nagoya University.
Completed the Nagoya University Graduate
School of Engineering. Obtained Ph.D. in
Bioengineering. Post-doctoral fellow at the
Developmental and Stem Cell Biology Program
of the Hospital for Sick Children in Toronto,
concurrently assigned for a while to Research
fellow at the Ontario Human iPS Cell Facility.
Assigned in March 2010 to the current position,
concurrently assigned in October 2010 to
Sakigake (PRESTO) Researcher of the Japan
Science and Technology Agency (JST).
Assistant Professor
Long-term and stable expression of transgene is
critical for a successful gene therapy. Human iPS cells
have been introduced GFP reporter gene, then
differentiated into Embryoid bodies (EBs), to monitor
the transgene expression.
Members
●
Assistant Professor
Akitsu Hotta
●
Technical Staff
Naoko Fujimoto
Noriko Sasakawa
Saya Shirai
●
Graduate Student (Doctoral program)
Hongmei Li
●
Secretary
Katsura Noda
Realization of cell transplant therapy with
the use iPS cells requires clearing many
hurdles, including unification of iPS cell
induction processes, quality control of
established cell lines, creation of iPS cells
having undergone gene therapy for the cells
having congenital disease, and prevention
of tumorigenesis following cell transplant.
At our laboratory, these issues have been
challenged by using the gene transfer
technology which have been employed in
the field of gene therapy and by regulating
the epigenetic gene control mechanisms.
Development of high-quality iPS
induction method through
regulation of intranuclear
environments
We paid close attention to partially
reprogrammed iPS cells in an attempt of
developing a method for establishment of
high quality and safety iPS cells in a well
reproducible manner. Comparison of these
partially reprogrammed iPS cells with ES
cells and high-quality iPS cells revealed
diverse differences in terms of intranuclear
chromosome structure and epigenetic
Quality of iPS cells differs depend on its generation method. We perform immunofluorescence staining to analyze
nuclear structure and localization of specific nuclear proteins in mouse iPS cells by a confocal microscopy.
24
New gene therapy strategy for
hemophilia with the use of iPS cells
If efficient gene therapy with the use of
iPS cells derived from patients having
congenital gene anomalies is possible, it will
contribute not only to clarify disease
features and the pathways for its
development and differentiation but also to
expand the possibility of cell transplant
therapy with iPS cells which are now being
developed with a goal of clinical
application.
For example, hemophilia A is a type of
congenital coagulopathy induced by
abnormality of clotting factor VIII (gene).
In cases of severe hemophilia A, the clotting
factor VIII activity is below a few percent of
the activity in healthy individuals, resulting
in difficulty in hemostasis. This disease can
be managed by factor replacement therapy,
which involves frequent injection of
expensive clotting factor preparations. The
necessity of repeating injection at intervals
of several days causes large physical and
economic burdens on the patients. For this
reason, development of a new method of
treatment for this disease is needed. If the
deficient clotting factor can be introduced
into the iPS cells derived from hemophilia
patients by means of gene transfer
technology and if the secretion of such a
factor can be kept at a high level after gene
transfer, there is the possibility that the
normal blood clotting activity is retained
for a long period of time. Considering the
knowledge that clotting factor VIII is
primarily produced in the liver in vivo, we
Research under way on a new iPS cell establishment method
through regulation of intranuclear environments and a method of
cell therapy using iPS cells
Assistant professor Akitsu Hotta, Ph.D. in Bioengineering, has been conducting
research in settings close to clinical practice from the beginning of his research career.
He is attempting to apply the gene transfer technology (employed for gene therapy)
and the methods for regulation of epigenetic control mechanism to establishment and
selection of iPS cells, with a goal of their application to cell transplant therapy.
During past studies, comparison of low-quality iPS cells (appearing during
establishment of iPS cells) with ES cells or high-quality iPS cells revealed differences in
intranuclear chromosome structure and epigenetic modifications. Thus, analysis of
these differences is expected to enable checking the quality of iPS cells. He also plans to
conduct research on artificially modifying the intranuclear structure to make the
intranuclear environments closer to those of ES cells, with a goal of developing a new
method of iPS cell creation.
At present, he is paying close attention to hemophilia A, a type of congenital gene
anomaly. He is carrying out also research on induction of differentiation of iPS cells into
the target type of cells and on expression of clotting factors, with a goal set at
development a new method of treatment by which secretion of the deficient clotting
factor in vivo in the patient can be achieved through transplant of patient-derived iPS
cells transfected with the gene for the clotting factor.
will explore cell types optimal for
formation of clotting factor VIII and
transplantation, and will establish a method
for induction of differentiation of such a
type of cell from iPS cells. To achieve high
expression of clotting factors for long term,
we have been developing virus vectors and
non-viral vectors, and analyzing the longterm stability of expression in human iPS
cells, using the EGFP reporter gene
expression as an indicator. We will apply
these techniques from now and develop
systems for prevention of the formation of
teratoma from transplanted iPS cells. In the
future, we will check the safety and efficacy
of each vector system developed so that it
can be used in clinical trials.
105
10 4
PE-GR-A
modifications. It was additionally shown
that conversion of low-quality iPS cells into
high-quality iPS cells by treatment with
specific reagents was accompanied by a
change in the nucleosome structure. These
differences in intranuclear chromosome
structure should serve as a hallmark of
successfully reprogrammed iPS cells. From
now, we plan to attempt manipulating the
intranuclear structure artificially with
several candidate factors, making use of
these changes in intranuclear structure as
landmarks. In the future, we will attempt
developing a new feasible method of iPS
cell derivation.
Furthermore, to select only highquality iPS cells during heterogeneous iPS
cell induction processes, we will modify the
human iPS cell selection and purification
methods, making use of genome
modification technology and the markers
specifically expressed on stem cells.
103
102
0
0
102
103
FITC-A
10 4
105
We introduced GFP (Green Fluorescent Protein) and
RFP (Red Fluorescent Protein) genes into iPS cells to
analyze transduction efficiency by a Flow
cytometer. Each color is well segregated.
Publications
1. Kattman SJ, Witty AD, Gagliardi M,
Dubois NC, Niapour M, Hotta A, Ellis
J, Keller G.
Stage-specific optimization of
Activin/Nodal and BMP signaling
promotes efficient cardiovascular
differentiation of mouse and human
pluripotent stem cell lines
Cell Stem Cell 8 (2): 2011. In press.
2. Hotta A, Cheung AY, Farra N, Garcha
K, Chang WY, Pasceri P, Stanford WL,
Ellis J.
EOS lentiviral vector selection system
for human induced pluripotent stem
cells.
Nature Protocols 4 (12):1828-1844,
2009.
3. Rastegar M, Hotta A, Pasceri P,
Makarem M, Cheung AY, Elliott S,
Park KJ, Adachi M, Jones FS, Clarke
ID, Dirks P, Ellis J.
MECP2 isoform-specific vectors with
regulated expression for Rett
syndrome gene therapy.
PLoS ONE 4 (8) : e6810, 2009.
4. Hotta A, Cheung AY, Farra N,
Vijayaragavan K, Seguin CA, Draper
JS, Pasceri P, Maksakova IA, Mager
DL, Rossant J, Bhatia M, Ellis J.
Isolation of human iPS cells using EOS
lentiviral vectors to select for
pluripotency.
Nature Methods 6 (5) : 370-37.6, 2009.
5. Hotta A, Ellis J.
Retroviral vector silencing during iPS
cell induction: an epigenetic beacon
that signals distinct pluripotent
states.
J Cell Biochem. 105 (4): 940-948, 2008.
25
Junya Toguchida
Profile
M.D., Ph.D.
Deputy Director, Department Head & Professor
Born in Yonago City in 1956. Graduated from
Kyoto University School of Medicine in 1981.
Completed the Kyoto University Graduate
School of Medicine in 1989. During the years of
graduate school, engaged in research on
anti-oncogenes. In 1995, appointed Associate
Professor at the Kyoto University Biomedical
Engineering Center, beginning research on
advancing medical engineering into
regenerative medicine, accompanied by clinical
services at the university hospital. Appointed to
Professor at the Institute for Frontier Medical
Sciences, Kyoto University in 2003, concurrently
assigned to Professor at CiRA in April 2010.
Fig. 1 Clinical study on bone regeneration therapy
with mesenchymal stem cells
Members
●
Professor
Junya Toguchida
●
Lecturer
Tomohisa Kato
●
Researchers
Makoto Ikeya
Jin Yonghui
●
Technical Staff
Yukiko Kobayashi
Sanae Nagata
Michka Hiraga
●
Graduate Students
Akira Nasu
Kazuo Hayakawa
Kyosuke Kobayashi
Sakura Tamaki
Elalaf Hassan
Yoshihisa Matsumoto
Koji Yokoyama
Takayuki Sawano
●
Secretaries
Hisayo Yasuda
Marie Yoshino
26
Research background: Research/
clinical practice on bone/soft tissue
sarcoma fused to research on
mesenchymal stem cells
I am now involved not only in the
CiRA but also at other units of Kyoto
University, with the primary role being
Professor in the field of tissue regeneration
and application at the Institute for Frontier
Medical Sciences (hereinafter abbreviated
as IFMS ). At the IFMS, I am undertaking
several studies pertaining to regeneration of
mesenchymal tissue, including studies for
elucidation and clinical application of the
essential biological nature of mesenchymal
stem cells (MSCs) which are stem cells
constituting the tissues and have the
potential of differentiating into tissues of
mesenchymal cell origin such as bone,
cartilage and fat. Particular emphasis has
been laid on research for implementation
of cell therapy with the use of MSC in
recent years. To implement cell therapy
with the use of stem cells, it is necessary to
comply with the provisions set forth in the
Guidelines on Clinical Studies Using
Human Stem Cells. This means the
necessity of the following multiple-step
processes: (1) cell isolation, culture and
quality assurance, (2) preclinical studies in
experimental animals, (3) validation at a
Cell Processing Center (CPC) of the GMP
(Good Manufacturing Practice: rules on
production and quality control) level, (4)
preparing a Product Overview, (5) devising
a plan on the basis of the Product
Overview, and (6) inspection and approval
at the institutional review board and the
Ministry of Health, Labour and Welfare
(MHLW) Ethics Committee. We began a
clinical study in 2008 after having followed
the above-mentioned processes. The study
we started is aimed at developing bone
regenerative therapy with the use of MSC,
assuming the form of a study jointly
conducted with the Department of
Orthopaedics and two CPSs, i.e., the Center
for Cell and Molecular Therapy (CCMT)
and the Translational Research Center, of
the Kyoto University Hospital (Fig. 1). The
interim results demonstrated the
effectiveness of this therapy. We plan to
advance this therapy to the next stage
(development of frontier healthcare
techniques on the basis of this therapy).
My second role pertains to diagnosis and
treatment as a clinician at the Department
of Orthopaedics of the Kyoto University
Hospital, covering tumors developing in
bone and soft tissue, particularly sarcoma
(a type of malignant tumor). My career as a
researcher started during research on
sarcoma at graduate school. Still at present,
I am involved in studies at IFMS (projects
on application of genetic diagnosis, and
identification of molecules serving as
targets of treatment) jointly with staff from
the university hospital. Since the years of a
graduate student, my greatest interest
pertains to the mechanism for development
of sarcoma, particularly sarcoma of a
cellular origin. Recently, a proposal was
made on the hypothesis that the essential
nature of tumor originates from a group of
cells called tumor stem cells. MSC has
thus begun to attract close attention as an
interface between oncology and stem cell
biology. As a result, the two fields of my
research at the IFMS are being fused to
each other.
Elucidation of bone-cartilage
diseases and creation of new drugs
for these diseases with the use of
iPS cells
Under the background illustrated
above, my research at the CiRA can be
roughly divided into two topics. One topic
is development of methods for induction of
iPS cell differentiation into mesenchymal
cells. The second topic is elucidation of the
features of intractable diseases and creation
of new drugs for treatment of these diseases
with the use of such methods. To put it
concretely, my research pertains to diseases
involving abnormal differentiation and
proliferation of bone and cartilage, i.e.,
fibrodysplasia ossificans progressive (FOP)
and CINCA (chronic inflammatory
neurological cutaneous articular)
syndrome. It is aimed at obtaining findings
useful in better understanding of these
diseases through establishment of iPS cells
from the somatic cells of patients with each
disease, subsequent induction of the iPS
cells differentiation into osseous cells or
chondrocytes and analysis of the
phenomena arising during these processes
in comparison to those of iPS cells from
healthy individuals. Methods for induction
of iPS cell differentiation into osseous cells
or chondrocytes are now close to
establishment (Fig. 2). Using these
methods, we will reproduce diseases in
vitro, with an ultimate goal of creating new
drugs with the use of a high throughput
screening system. Interestingly, the
progression of each research has been
triggered or accelerated by the findings
from studies on malignant tumors
(osteosarcoma and chondrosarcoma), and I
think that this may be one of the interfaces
between cancer and regenerative medicine.
Another task is to put the Facility for
iPS Cell Therapy (FiT), a CPC of the CiRA,
into full operation as a facility capable of
preparing clinically applicable cells, so that
cell therapy with the use of iPS cells can be
smoothly implemented in the future. As a
concrete action towards this goal, we plan
to utilize FiT as a place for preparing the
cells used in the clinical study on MSC
currently under way at the CCMT (a CPC
of the Kyoto University Hospital). To this
end, we are arranging adequate
environments for FIT, transferring the
Attempting realization of bone/cartilage
cell therapy as an orthopedic surgeon
Prof. Junya Toguchida is concurrently Professor at the Department of Tissue
Regeneration, the Institute for Frontier Medical Sciences, Kyoto University. He also
serves as a clinician at the Department of Orthopaedic Surgery, Kyoto University
Hospital. Since the years of a graduate student, he has been engaged in research on the
mechanism for onset of sarcoma, attempting to elucidate the essential nature of
mesenchymal stem cells (MSCs) capable of differentiating into tissues of mesenchymal
cell origin such as bone, cartilage and fat and to apply the findings to cell therapy. In
2008, he began a clinical study on bone regeneration therapy at the same university
hospital.
At the CiRA, research is under way with the goal of establishing disease-specific iPS
cells (i.e., iPS cells specific to fibrodysplasia ossificans progressive and CINCA syndrome),
to induce differentiation of these cells into osseous cells and chondrocytes, to elucidate
the features of these diseases through comparison with iPS cells from healthy individuals
and to create new drugs for the treatment of these diseases.
He is also involved in operation of the Facility for iPS Cell Therapy (FiT) which is an
iPS cell processing facility. Modeling after the ways used for preparing the cells for the
clinical trial on MSC currently under way at the same university hospital, Prof. Toguchida
will take further steps related to arrangement of the environments and the procedure
for research and the education/training for staff and will begin preparing cells of the
level applicable to clinical studies within fiscal 2011.
Fig. 2 Induction of differentiation from iPS cells
From bone marrow stromal cells (A), iPS cells (B) were
established, followed by induction of differentiation
into osseous cells (C) and chondrocytes (D).
CCMT s SOP (Standard Operating
Procedure) to FiT, and are taken
preparatory steps to start GMP level cell
production at FiT within fiscal 2011.
As illustrated above, my research
pertains to diverse fields, but all of these
fields are united with some key words. I
believe that we can contribute to
advancing research on each field through
organic linkage among these fields. I
hope participation of researchers full of
volition in this laboratory.
Publications
1. Nishigaki T, Teramura Y, Nasu A,
Takada K, Toguchida J, Iwata H.
Highly efficient cryopreservation of
human induced pluripotent stem
cells using a dimethyl sulfoxide-free
solution.
Int J Dev Biol. 2011. in press.
2. Aoyama T, Okamoto T, Fukiage K,
Otsuka S, Furu M, Ito K, Jin Y, Ueda
M, Nagayama S, Nakayama T,
Nakamura T, Toguchida J.
Histone modifiers, YY1 and p300,
regulate the expression of cartilagespecific gene, chondromodulin-I, in
mesenchymal stem cells.
J Biol Chem. 285(39): 29842-29850,
2010.
3. Ito K, Aoyama T, Fukiage K, Otsuka
S, Furu M, Jin Y, Nasu A, Ueda M,
Kasai Y, Ashihara E, Kimura S,
Maekawa T, Kobayashi A, Yoshida S,
Niwa H, Otsuka T, Nakamura T,
Toguchida J.
A novel method to isolate
mesenchymal stem cells from bone
marrow in a closed system using a
device made by non-woven fabric.
Tissue Eng Part C Methods. 16(1):
81-91, 2010.
27
Jun Takahashi
Profile
M.D., Ph.D.
Associate Professor
Born in Itami, Hyogo in 1961. Graduated from
Kyoto University School of Medicine. Completed
the Kyoto University Graduate School of
Medicine. Acquired PhD in Medicine. Joined the
Department of Neurosurgery, Kyoto University,
and certified as neurosurgeon in 1993. Studied
at Salk Institute, USA (Dr. Fred Gage s
Laboratory) for 2 years from 1995. Appointed
Associate Professor at the Institute for Frontier
Medical Sciences, Kyoto University in 1997.
Concurrently assigned to Associate Professor at
the Center for iPS Cell Research and Application,
Kyoto University since 2008.
Fig. 1 Induction of dopaminergic neurons from
human iPS cells by suspension culture. Dispersed
human iPS cells are subjected to suspension culture
to induce their differentiation into neurons. If the
cells are subsequently subjected to adhesion culture,
they extend neurites. Green cells are Tuj1 (protein
specifically expressed on postmitotic neurons)
positive neurons. Red cells are TH (tyrosine
hydroxylase, a marker for dopaminergic neurons)
positive dopaminergic neurons.
Members
●
Associate Professor
Jun Takahashi
●
Researchers
Asuka Morizane
Daisuke Doi
Kaneyasu Nishimura
●
Technical Staff
Kei Kubota
Mitsuko Katsukawa
Makoto Motono
Emi Yamasaki
●
Graduate Students
Masanori Gomi
Kazuo Washida
Tetsuhiro Kikuchi
Akihiro Kitamura
Tatsuya Yoshikawa
Aya Ogura
Bunpei Samata
●
We have been conducting research with a
goal set at realization of neuroregenerative
medicine with the use of iPS cells. The
target disease is Parkinson s disease, an
intractable progressive neurological disease.
In patients with this disease, loss of
dopaminergic neurons projected from the
substantia nigra in the midbrain to the
striatum reduces the intracerebral
dopamine level, resulting in physical
rigidity, difficulty in movement and
tremor. We attempt to induce
dopaminergic neurons from iPS cells so
that the lost dopaminergic neurons can be
replenished by transplantation of the
induced cells into the brain. Clinical
application of cell replacement therapy
with the use of iPS cells needs to resolve
several open issues listed below.
(1) Safe and efficient induction of
differentiation into neurons
In the attempts reported to date, mouse
feeder cells were used to induce
dopaminergic cells from human ES cells or
iPS cells. In clinical cases, however, it is not
possible to use mouse cell as a rule. For this
reason, we have developed a technique for
efficient induction of nerve cells without
using feeder cells and have reported this
technique as an outcome from the current
year s research at our laboratory.
iPS have the potentials of
differentiating into various cells. If it is
attempted to induce only one type of cell
(nerve cells in our case) from iPS cells, it is
necessary to suppress the differentiation of
iPS cells into the other types of cell. For this
purpose, we are using two low-molecule
compounds. One is dorsomorphin (a BMP
signal inhibitor) and the other is SB431542
Undergraduate Student
Yusuke Nakajima
●
Secretary
Toshiko Gomibuchi
Fig. 2 Synchronous grafting of mouse ES cell-derived neural progenitorcells and matrigel into the brain. Four
weeks after grafting, immunostaining reveals increase in the graft size and the number of TH positive surviving
cells (red) as compared to the uncombined cell grafting group.
28
Research on cell replacement therapy with iPS cells for Parkinson s disease
Parkinson s disease is a progressive neurological disease causing tremor and dyskinesia.
This disease has been primarily attributed to reduction in dopamine level due to loss of
neurons secreting the neurotransmitter dopamine in the brain. At the Takahashi
Laboratory, research is under way to develop methods of inducing dopaminergic neurons
from iPS cells and to transplant these cells into the brain.
Mouse-derived feeder cells, often used for culture to induce differentiation of
human iPS cells, are inappropriate for clinical application. For this reason, they have
developed a technique enabling efficient induction of neurons from human iPS cells
without using feeder cells. Furthermore, in an attempt of facilitating differentiation into
neurons alone, they have succeeded in improving the efficiency of induction by
combining two methods, i.e., the method of inhibiting cell differentiation signals using
low-molecule compounds and the method of incubating dispersed iPS cells on a U-form
bottom dish to induce formation of neurospheres. They have additionally found and
reported that grafting of matrigel (extracellular matrix) together with the cells resulted in
a higher survival rate of dopaminergic neurons in the recipient, possibly suppressing
inflammation. They are conducting quantitative evaluation of exercise level, PET-CT and
so on using monkey models of diseases treated with ES cells.
Fig. 3 Relationship between the findings from
[11C]-CFT PET CT and the video-based quantitative
evaluation of spontaneous movoments. In a monkey
model of Parkinson s disease, i.e., monkeys treated
with MPTP (1-methyl-4-phenyl-1,2,3,6tetrahydropyridine) known to induce Parkinsonismlike symptoms, a positive correlation was noted
between midbrain dopaminergic function and
spontaneous movements.
(a TGFβ/Activin/Nodal signal inhibitor).
Inhibition of these two signals suppresses
the self-proliferation of iPS cells and their
differentiation into muscles and internal
organs, leading to stimulation of
differentiation into neural cells. The
efficiency of inducing neurons from human
ES and iPS cells varies depending on the
cell line used. However, our technique
enables induction of cells of the nervous
system at an efficiency of about 100%
(from most cells used) with each cell line.
We have additionally developed a technique
by which iPS are initially cultured in
dispersed form in a U-shape bottom dish,
to stimulate differentiation into neurons
through formation of neurospheres.
Combining this culture method with the
two low-molecule compounds has made it
possible for us to induce neurons efficiently
without using feeder cells (Fig. 1).
(2) Improving the host s brain
environments
So that the cells grafted can survive
efficiently, it is important to optimize the
host s brain environments. Puncture of the
brain with a needle for transplantation
causes inflammation in the brain,
accompanied by immune reactions in case
of allografting. We previously
demonstrated that inflammation and
immune reactions suppress the
differentiation of grafted cells intoneurons.
During the current year, we showed that
grafting of matrigel (basement membrane
matrix) with cells resulted in more survival
of dopaminergic neurons (Fig. 2). In this
case, matrigel is considered to play not only
the role of scaffold for survival of grafted
cells but also the role of preventing the
inflammatory cell s attack on grafted cells
physically and supplying trophic factors.
(3) Analysis using primate models
of diseases
Mice and rats, which are often used in
animal studies, differ from humans in
terms of size and neurological anatomy. In
view of clinical application, experiments
using primates seem to be essential during
preclinical studies. We have been
conducting experiments on transplantation
of ES cells using a crab-eating monkey
(Macaca fascicularis) model of
Parkinson s disease. In those experiments,
scoring was employed for evaluation of
behaviors (hand/foot tremor, stability
during walk, and so on). For more
objective evaluation, we used video-based
quantitative evaluation of the active
exercise level and reported its results as
the outcome of the current year s
research. The results from such videobased analysis correlated also with the
results of conventional scoring-based
evaluation and the dopaminergic
function assessed by positron emissionCT (PET-CT), suggesting that videobased analysis will be useful in preclinical
studies from now on (Fig. 3).
On the basis of these results, we are
further optimizing the settings as to the
three aspects listed above. In the near
future, we will confirm the efficacy and
safety of this approach in preclinical
studies using human iPS cells and
primate models of diseases.
Publications
1. Morizane A, Doi D, Kikuchi T, Nishimura K, Takahashi J.
Small molecule inhibitors of BMP and Activin/Nodal signals promote highly
efficient neural induction from human pluripotent stem cells.
J Neurosci Res.2010. published online
2. Saiki H, Hayashi T, Takahashi R, Takahashi J.
Objective and quantitative evaluation of motor function in a monkey model of
Parkinson s disease.
J Neurosci Methods 190: 198-204, 2010.
3. Uemura M, Refaat MM, Shinoyama M, Hayashi H, Hashimoto N, Takahashi J.
Matrigel supports survival and neuronal differentiation of grafted embryonic
stem cell-derived neural precursor cells.
J Neurosci Res. 88: 542-551, 2010.
4. Hayashi H, Morizane A, Koyanagi M, Ono Y, Sasai Y, Hashimoto N, Takahashi J.
Meningeal cells induce dopaminergic neurons from embryonic stem cells.
Eur J Neurosci. 27: 261-268, 2008.
29
Jun K.Yamashita
Profile
M.D., Ph.D.
Associate Professor
Born in Kyoto City in 1965. Graduated from
Acquired PhD in Medicine in 1998. In 2003,
Associate Professor (PI) at the Department of
Stem Cell Differentiation, Institute for Frontier
Medical Sciences, Kyoto University. In 2008,
concurrently assigned to Associate Professor at
the Center for iPS Cell Research and Application,
the Institute for Cell-Material Sciences, Kyoto
University. Current position since 2010. During
the years of graduate student, engaged in
analysis of molecular mechanisms for vascular
proliferative diseases at the Department of
Medicine and Clinical Science (Prof. Kazuwa
Nakao) and later began research on ES cells at
the Department of Molecular Genetics (Prof.
Shin-ichi Nishikawa). At present, conducting
comprehensive research, focusing on
cardiovascular differentiation and regeneration
with the use of ES cells and iPS cells.
Fig. 1 Cardiomyocytes undergoing proliferation
following drug treatment. Drug treatment resulted
in uptake of large amounts of EdU into the nuclei
(DNA replication; green) of the purified
cardiomyocytes (red) induced from ES cells.
Members
●
Associate Professor
Jun K. Yamashita
●
Researchers
Masafumi Takeda
Kohei Yamamizu
Masataka Fujiwara
Takuhiro Hoshino
●
Technical Staff
Mizuho Shino
Shiori Katayama
●
Graduate Students
Hideki Uosaki
Genta Narazaki
Taichi Matsunaga
Kent Doi
Hidetoshi Masumoto
Takehiko Matsuo
Hiroyuki Fukushima
Hiromi Kumamoto
●
Research Student
Yajing Liu
●
Secretary
Chisato Murayama
30
During the current year, researches with
various approaches were undertaken about
the five topics listed below.
1. Induction of cardiomyocyte
differentiation from ES and iPS cells
1) Control of Cardiomyocyte proliferation:
Cardiomyocytes cease growth soon after
differentiation but its mechanism remains
unexplained. To elucidate the mechanism
for suppression of cardiomyocyte growth
and to control that, we screened small
molecules capable of inducing
cardiomyocytes proliferation with the use
of ES-derived early-stage cardiomyocytes.
To date, we have identified 4 compounds
capable of regulating different signal
pathways. Using appropriate combinations
of compounds, we have achieved 14-fold
increase at maximum in the number of
purified cardiomyocytes (paper submitted;
patent pending) (Fig. 1).
2) Identification of new substances capable
of stimulating cardiomyocyte
differentiation: We are screening substances
capable of stimulating differentiation into
cardiomyocytes among the chemical library
aiming at efficient induction of
cardiomyocyte differentiation and utilizing
them as drugs for cardiac regeneration in
the future (joint research with Waseda
University). Last year, we demonstrated
that cyclosporin A (CSA), an
immunosuppressant, has potent activity in
inducing differentiation into
cardiomyocytes, elevating the efficiency of
differentiation about 10-fold (Yan, Biochem
Biophys Res Commun., 2009; patent
pending). Recently, we identified a
substance more potently inducing the
differentiation at a concentration as low as
1/1000 of the CSA level.
3) Development of efficient methods for
induction of human iPS cell differentiation
into cardiomyocytes: On the basis of these
outcomes from previous studies, we have
been developing new methods for efficient
induction of differentiation. We have
shown that application of the CSA method
to induction of human iPS cell
differentiation by the END2 cell method
(Mummery, Circulation, 2003) elevated the
efficiency of human iPS cell differentiation
into cardiomyocytes about 4-fold as
compared to the conventional efficiency.
The thus induced human myocardial cells
showed various responses (changes in heart
rate, QT prolongation, etc.) to drugs,
suggesting their applicability as a human
cardiomyocyte model (Fujiwara, PLoS One,
2011).
2. New strategy of cell therapy to
achieve cardiac regeneration
Development of cell transplantation
technique with cardiac tissue sheets: We
have developed a new technique for
systematic induction of cardiovascular cells
(vascular endothelial cells, vascular mural
cells, cardiomyocytes, blood cells, etc.) from
mouse ES and iPS cells (Yamashita, Nature,
2000; FASEB J, 2005; Narazaki, Circulation,
2008, and others). At present, we are
developing new techniques of cell
transplantation through combining cells
induced from ES/iPS cells with the cell
sheet technology (with the use of
temperature-sensitive culture dish; joint
research with Tokyo Women s Medical
University). We observed significant
improvement of cardiac function when the
pulsatile cardiac tissue sheet made of ES
cell-derived cells was transplanted to a rat
myocardial infarction model (joint research
with the Department of Cardiovascular
Surgery; paper in preparation) (Fig. 2).
3. Analysis of the mechanism for
vascular diversification
1) Elucidation of the mechanism for
arterial/venous differentiation: Following
success in inducing the differentiation into
three types of endothelial cells (arterial,
venous and lymph duct endothelial cells)
(Yurugi-Kobayashi, Arterioscler Thromb
Vasc Biol., 2006; Kono, Arterioscler Thromb
Vasc Biol., 2006), we have conducted more
detailed analysis of the molecular
mechanism for differentiation into arterial/
venous endothelial cells, revealing a
previously unknown molecular mechanism
by which arterial endothelial cells are
induced through simultaneous activation
of Notch and β-catenin signals mediated
by PI-3 kinase under cAMP signaling
(Yamamizu, J Cell Biol., 2010).
2) Elucidation of mechanisms for cAMP/
PKA-induced stimulation of endothelial
differentiation: Following our previous
discovery of cAMP signal-induced
stimulation of the differentiation of
vascular precursor cells (F1kl positive cells)
into endothelium, we found that PKA
(protein kinase A), located downstream of
cAMP, increases the expression of F1k1 and
neuropilin 1, which form a sensitive
receptor for VEGF (vascular endothelial
growth factor), resulting in enhanced
reactivity of vascular progenitor cells to
VEGF (Yamamizu, Blood, 2009).
4. Analysis of basic molecular
mechanisms for early stage
differentiation of pluripotent stem
cells
We are now analyzing the molecular
mechanisms, especially epigenetic
regulation during early differentiation of
undifferentiated ES cells.
5. Development of new basic tools
for research on stem cell
differentiation
Gene knockdown-rescue ES/iPS cell
system: We are developing a cell system
capable of suppression and stimulation
of the expression of target genes with a
combination of inducible shRNA (or
microRNA) and inducible cDNA
expression systems.
Extensive research involving genes, molecules, cells and tissues
with a goal of application to proliferation/differentiation of
cardiomyocytes and treatment of diseases
At the Yamashita Laboratory, research aimed at establishing cardiac regenerative therapy
had been conducted, involving elucidation of the mechanisms for early arresting of
myocardial growth and exploration of the methods for control of cardiomyocyte
proliferation and differentiation. To date, they have found compounds enhancing the
proliferation of cardiomyocytes derived from ES cells. In addition, through screening of
compounds having the potential of stimulating cardiomyocyte differentiation, they have
found and reported that cyclosporin A (CSA), an immunosuppressant, can serve as a
candidate compound for such a purpose. These results have been applied to induction
of human iPS cell differentiation into cardiomyocytes, resulting in improved efficiency of
differentiation.
Furthermore, they have developed techniques for systematic induction of vascular
cells, cardiomyocytes and blood cells from mouse ES cells and iPS cells and are trying to
explore a novel cardiac regenerative strategy with a sheet of mouse ES cell-derived
cardiovascular cells.
They have been additionally engaged in elucidation of the molecular mechanisms for
vascular cell differentiation and diversification into arteries and vein, leading to discovery
of new signals and drugs capable of regulating such signals.
Cellular tools capable of suppressing and stimulating the expression of genes
involved in ES and iPS cell differentiation are also under development.
Fig. 2 A cardiac tissue sheet with mouse ES cellderived cardiovascular cells. Prepared by re-culture
of cardiovascular cells induced from mouse ES cells in
a temperature-sensitive culture dish. Pulsation is
macroscopically visible.
Publications
1. Fujiwara M, Yan P, Otsuji TG, Narazaki
G, Uosaki H, Fukushima H, Matsuda H,
Kuwahara K, Harada M, Matsuoka S,
Okita K, Takahashi K, Nakagawa M,
Ikeda T, Sakata R, Mummery CL,
Nakatsuji N, Yamanaka S, Nakao K,
Yamashita JK.
Induction and enhancement of cardiac
cell differentiation from mouse and
human induced pluripotent stem cells
with cyclosporine-A.
PLoS ONE 6:e16734, 2011.
2. Yamamizu K, Matsunaga T, Uosaki H,
Fukushima H, Katayama S, HiraokaKanie M, Mitani K, Yamashita JK.
Convergence of Notch and β -catenin
signaling induces arterial fate in
vascular progenitors.
J Cell Biol. 189:325-338, 2010.
3. Yamamizu K, Kawasaki K, Katayama S,
Watabe T, Yamashita JK.
Enhancement of vascular progenitor
potential by protein kinase A through
dual induction of Flk-1 and
Neuropilin-1.
Blood 114:3707-3716, 2009.
4. Yan P, Nagasawa A, Uosaki H,
Sugimoto A, Yamamizu K, Teranishi M,
Matsuda H, Matsuoka S, Ikeda T,
Komeda M, Sakata R, Yamashita JK.
Cyclosporin-A potently induces highly
cardiogenic progenitors from
embryonic stem cells.
Biochem Biophys Res Commun.
379:115-120, 2009.
5. Narazaki G, Uosaki M, Teranishi M,
Okita K, Kim B, Matsuoka S, Yamanaka
S, Yamashita JK.
Directed and systematic differentiation
of cardiovascular cells from mouse
induced pluripotent stem cells.
Circulation 118:498-506, 2008.
31
Kenji Osafune
Profile
M.D., Ph.D.
Associate Professor
Born in Tatsuno City, Hyogo Prefecture in 1971.
Graduated from Faculty of Medicine, Kyoto
University. Began to serve as nephrologist.
Completed the doctoral course at Graduate
School of Science, the University of Tokyo.
Acquired PhD in Science. Research on kidney
development and regeneration at the University
of Tokyo (Laboratory of Prof. Makoto Asashima)
from 2000 through 2005. Research on pancreas
regeneration with the use of human ES and iPS
cells at Harvard Stem Cell Institute/Department
of Stem Cell and Regenerative Biology, Harvard
University (Laboratory of Prof. Douglas A.
Melton) from 2005 through 2008. Current
position since 2008.
Fig. 1 Pancreatic precursor cells induced from human
iPS cells
Members
●
Associate Professor
Kenji Osafune
●
Assistant Professor
Taro Toyoda
●
Researchers
Toshikazu Araoka
Takafumi Toyohara
Maki Kotaka
Michinori Funato
Tetsuhiko Yasuno
●
Technical Staff
Sayaka Arai
Nanaka Nishimura
Tomomi Sudo
Yuko Kurose
●
Graduate Students
Fumihiko Shiota
Shin-Ichi Mae
Yasushi Kondo
●
Secretary
Erika Moriguchi
32
Group s goal: Development of
regeneration therapy and new
drugs for the treatment of
intractable diseases of kidney,
pancreas and liver
Intractable diseases of kidney, pancreas
and liver such as chronic kidney disease,
diabetes mellitus and liver failure are still
serious global issues not only from medical
points of view but also from
medicoeconomic viewpoints. Although
kidney transplantation, islet
transplantation and liver transplantation
are radical therapeutic methods for these
intractable disorders, serious shortage of
donor organs has been hampering these
approaches of treatment. To resolve this
problem, our research group has been
attempting to achieve regeneration of
kidney, pancreas and liver in vitro from iPS
cells, with a goal of creating cells applicable
to transplantation and developing new
drugs for the treatment of these disorders.
On the basis of the findings from studies in
the field of developmental biology, we are
developing methods for efficient induction
of iPS cell differentiation into cells
constituting these three organs. To this end,
we are attempting induction of
differentiation with the use of growth
factors and conducting High-Throughput
Screening (HTS) of low-molecular-weight
compounds capable of inducing the
differentiation of these cells. Using these
differentiation systems, we aim at: (1)
analysis of human developmental biology,
(2) development of cell transplantation
therapy, (3) creation of new models of
intractable diseases, and (4) development
of new drugs for treatment, and so on.
Progress report for the current year
1. Establishment of low-molecular-weight
compound screening systems
In October of this year, the HTS
Option (a product of Becton Dickinson
(BD)) was introduced to our laboratory
and fitted to the previously installed flow
cytometer FACS Fortessa. Thus, a highspeed screening system for low-molecularweight compounds with the use of flow
cytometer has been established. Using this
system, we have started screening of lowmolecular-weight compounds capable of
inducing the differentiation of human ES/
iPS cells into intermediate mesoderm (the
early embryonic tissue having the potential
to develop into kidney). Furthermore, a
HTS system based on image analysis with
immunostaining and an image analyzer
(InCell Analyzer 2000, GE Healthcare) has
been established, and we have started
screening of compounds inducing the
differentiation of human ES/iPS cells into
endoderm.
2. Kidney regeneration
We have started developing methods
for inducing the differentiation of human
and mouse ES/iPS cells into intermediate
mesoderm primarily with a combination of
growth factors.
3. Pancreas regeneration
The conventional methods for
inducing the differentiation of human ES/
iPS cells into definitive endoderm (the early
embryonic tissue retaining the potential to
differentiate into pancreas and liver) were
modified to develop methods enabling
induction of differentiation into endoderm
at an efficiency over 70% from human iPS
cell lines (201B6, 201B7, 253G1 and 253G4)
previously established at the Center for iPS
Cell Research and Application (CiRA),
Kyoto University as well as human ES cell
lines (H9, KhES1 and KhES3). By
combining these methods with existing
ones, we have succeeded in inducing
differentiation into pancreatic precursor
cells at an efficiency of about 50% (Fig. 1).
4. Liver regeneration
We have established methods for
inducing the differentiation of human
iPS cells into liver cells via hepatic
precursor cells at an efficiency of about
30%, by utilizing the above-mentioned
techniques for efficient differentiation of
human ES/iPS cells into endoderm (Fig.
2).
5. Creation of new disease models in vitro
using disease-specific iPS cells
Disease-specific iPS cells have been
established from 7 patients with autosomal
dominant polycystic kidney disease
Fig. 2 Liver cells induced from human iPS cells
(ADPKD) which is an intractable
hereditary kidney disease involving cyst
formation in many organs (kidney, etc.)
and can lead to end stage renal failure due
to cyst-caused destruction of renal
structure (Fig. 3). In 3 of these 7 cases,
mutation of the causative gene responsible
for this disease has been identified, and we
have begun the attempt of inducing the
differentiation of these ADPKD-specific iPS
cells into cells constituting the organs
affected by this disease. Furthermore, we
have completed skin biopsy from 3 patients
with microscopic polyangiitis (an
intractable disease of the category
vasculitis syndrome" causing
inflammation of the blood vessels of
systemic organs such as kidneys) and have
started an attempt of establishing iPS cells
specific to this disorder.
Generation of transplantable cells and development of new drugs
for the treatment of intractable kidney/pancreas/liver diseases
Associate Professor Kenji Osafune, having been engaged as an internist in the
management of intractable diseases of kidney, pancreas and liver, is keenly aware of the
necessity of regenerative therapy for these organs and is exploring growth factors and
chemical compounds capable of increasing the efficiency of differentiation of ES and
iPS cells on the basis of his research on organ regeneration in vitro.
During the current year, his laboratory established systems for screening of
compounds capable of inducing the differentiation of human ES and iPS cells into the
three organs with the use of flow cytometry and image analysis and began operation of
these systems.
Furthermore, the laboratory is developing or modifying the methods for induction
of pancreas and liver cells from human and mouse ES/iPS cells. Regarding pancreas,
among others, it has succeeded in achieving induction of endoderm at an efficiency
over 70% and subsequent differentiation into pancreatic precursor cells at an efficiency
of about 50%. Disease-specific iPS cells have been established from patients with
autosomal dominant polycystic kidney disease or vasculitis syndrome, and these cells
have begun to be used for induction of kidney cells.
Fig. 3 Disease-specific iPS cells established from the
somatic cells of patients with autosomal dominant
polycystic kidney disease
Publications
1. Osafune K. In vitro regeneration of kidney from
Exp.Cell Res. 316(16) : 2571-2577, 2010.
2. Lau F, Ahfeldt T, Osafune K, Akutsu
H, Cowan CA. Induced pluripotent stem (iPS) cells:
an up-to-the-minute review. F1000 Biology Reports 1: 84, 2009.
3. Chen S, Borowiak M, Fox J, Maehr R,
Osafune K, Davidow L, Lam K, Peng
L, Schreiber S, Rubin L, Melton DA. A small molecule that directs
differentiation of human embryonic
stem cells into the pancreatic lineage.
Nature Chem Biol.5(4):258-265, 2009.
4. Huangfu D, Osafune K, Maehr R, Guo
W, Eijkelenboom A, Chen S,
Muhlestein W, Melton DA. Induction of pluripotent stem cells
from primary human fibroblasts with
only Oct4 and Sox2.
Nature Biotechnol. 26(11):1269-75,
2008.
5. Osafune K, Caron L, Borowiak M,
Martinez RJ, Fitz-Gerald CS, Sato Y,
Cowan CA, Chien KR, Melton DA. Marked differences in differentiation
propensity among human embryonic
stem cell lines. Nature Biotechnol.
26(3):313-5, 2008.
33
Department of Clinical Application
Tatsutoshi Nakahata
Profile
M.D., Ph.D.
Deputy Director, Department Head & Professor
Members
●
Professor
Tatsutoshi Nakahata
●
Assistant Professor
Megumu Saito
●
Researchers
Akira Niwa
Koichi Oshima
Munetada Haruyama
●
Technical Staff
Yuko Sasaki
Mayu Yamane
Yukari Shima
Shota Tomita
Hiroshi Koyanagi
●
Graduate Students
Takayuki Tanaka
Itaru Kato
Masakatsu Yanagimachi
At our laboratory, research has been
conducted to establish iPS cells from
patients with diverse diseases and to
analyze the features and etiology of various
diseases using these cells, with a goal of
applying iPS cells to clinical studies. For
efficient research on disease-specific iPS
cells, it is necessary to establish and
combine the following three experimental
systems appropriately: (1) establishment of
disease-specific iPS cells, (2) creation of an
optimal differentiation system capable of
reflecting the features of disease, and (3)
analysis of differentiation-induced cells. We
have been conducting research, with close
attention primarily paid to hematological
diseases (dyshematopoiesis, etc.),
immunodeficiency, intractable pediatric
neurological diseases, congenital hearing
loss1 and congenital muscular diseases2 ,
and have been analyzing these diseases.
Yoshinori Sugimine
Takehiro Iki
Michiko Yoshida
Naoya Maekawa
●
Secretary
Harumi Watanabe
34
1．Establishment of diseasespecific iPS cells
At our laboratory, iPS cells have been
established from more than 20 patients
with 8 kinds of hematological,
immunological or neurological diseases or
their family members to date (including the
cells now being established)3. These diseases
include diseases for which establishment of
iPS cells are very difficult because of
disease-specific biological features.
Born in Komae City, Tokyo in 1945. Graduated
from Shinshu University School of Medicine in
1970. After services at Showa Inami General
Hospital, Kofu City Hospital and National Toshin
Hospital, assigned to Teaching Assistant at
Shinshu University Hospital (Department of
Pediatrics) and then Chief Physician at
Department of Pediatrics, Iida City Hospital.
Research Fellow at University of South Carolina,
USA, for 2 years since 1980. Teaching Assistant,
Lecturer and Associate Professor at Shinshu
University School of Medicine since 1983.
Professor at Department of Clinical
Oncology , Institute of Medical Science,
University of Tokyo in 1993. Professor at
Department of Pediatrics, Graduate
School of Medicine, Kyoto University in 1999.
Professor and Vice Director at the Center for iPS
Cell Research and Application, Institute for
Integrated Cell-Material Science, Kyoto
University in 2009. Current position since 2010.
Establishing iPS cells specific to these
diseases is expected to contribute to
elucidating the mechanism for
reprogramming, and we are making various
attempts to facilitate establishment of iPS
cells. The established iPS cells have been
analyzed as to expression of
undifferentiated markers, silencing of
introduced genes, potentials of forming
teratoma, and karyotype, followed by
selection of appropriate clones. Our
laboratory has been coordinating the
research on disease-specific iPS cells at
Kyoto University and working in
cooperation with other iPS cell research
centers of the Ministry of Education,
Culture, Sports, Science and Technology
(MEXT) to facilitate active deposition of
established iPS cells to the RIKEN
BioResource Center.
2．Creation of differentiation
systems
To enable high quality analyses,
creation of appropriate differentiation
systems is indispensable. Our laboratory
has been engaged primarily in modifying
the blood cell differentiation systems and
developing unique systems which enable
appropriate tracing of the differentiation
pathways in the living body. If these blood
cell differentiation systems are employed, a
variety of functional blood cells (e.g., red
blood cells, neutrophils and platelets) can
be prepared. We are using these symptoms
for analysis of diseases. Furthermore, we
are conducting research, which involves
introducing the blood cells differentiated in
vitro into immunodeficient mice (e.g.,
NOG mice developed at our laboratory)
and subsequently reconstructing the blood
cells system in vivo.
3．Analysis of differentiated cells
The use of disease-specific iPS cells
enables analysis of multiples types of cell
and tissue associated with a single disease
in a cross-sectional manner, revealing the
entire profile of the disease. In this
connection, we are focusing on congenital
pediatric diseases. Congenital pediatric
diseases often involve abnormalities of
multiple tissues, e.g., abnormalities of
blood cells + cartilage, and blood cells +
nervous system. Thus, comprehensive
analysis of these diseases with the use of
disease-specific iPS cells is very useful in
understanding the disease concerned. To
date, we have succeeded in reproducing the
features of several diseases in vitro. From
now, we will further promote research in
this field to facilitate elucidation of the
etiology and features of these diseases.
Furthermore, since disease-specific iPS cells
are considered to be very useful as a tool for
creating drugs for the treatment of
intractable diseases, we will carry out
screening of drugs and compounds which
deserve phenotype analysis in vitro, with a
goal of contributing to management of
patients suffering from intractable disease.
Perspectives for the coming and
subsequent years
Patient-derived iPS cells are useful in
clarifying the features at the cellular level of
diseases known to involve hereditary
Derivation of blood cells from human iPS cells from
human iPS cells
Research on etiology and treatment of pediatric hematological, immunological
and neurological diseases with the use of disease-specific iPS cells
Prof. Tatsutoshi Nakahata, also serving as a pediatrician, has been long engaged in the
treatment of intractable pediatric diseases. At present, he is conducting research for analysis
of etiology and features of such diseases with the use of disease-specific iPS cells. The
diseases covered by his research include hematological diseases (dyshematopoiesis, etc.),
immunodeficiency, intractable pediatric neurological diseases, congenital hearing loss and
congenital muscular diseases. To date, he has established iPS cells from more than 20 patients
with 8 kinds of disease or their family members. He is additionally conducting research
involving transplantation of established disease-specific iPS cells into immunodeficient mice to
achieve their survival and tissue reconstruction. He also plans to use these cells for screening
of drugs/compounds and as a drug creation tool. It is also a goal of his research to utilize the
findings from these studies in elucidating the mechanism for cell reprogramming.
As an essential step for development of a library of disease-specific iPS cells, the
Nakahata Laboratory has been coordinating research on disease-specific iPS cell lines at Kyoto
University and assuming the role of promoter for deposition of established iPS cells to the
RIKEN BioResource Center.
factors in the etiology. If various somatic
cells of patients carrying unidentified
hereditary factor in the etiology are made
into disease-specific iPS cells, they will
facilitate understanding of interactions
among cells and environmental factors
affecting the formation of etiological
factors for diverse diseases. They are also
useful in evaluating the safety of drugs and
exploring new drugs for treatment of
diseases. Thus, patient-derived iPS cells
combined with gene therapy are expected
to provide a means of cell transplant
therapy tailored to individual patients.
1．Joint research with Dr. Juichi Ito (Department of
Otorhinolaryngology,Kyoto University Graduate
School of Medicine)
2. Joint research with Dr. Toshio Heike (Department
of Developmental Pediatrics, Kyoto University
Graduate School of Medicine)
3. Including joint research with Associate Prof. Isao
Asaka, Associate Prof.Haruhisa Inoue and Lecturer
Keisuke Okita (Center for iPS Cell Research
andApplication, Kyoto University)
Publications
1. Mizuno Y, Chang H, Umeda K, Niwa A, Iwasa T, Awaya T, Fukada SI, Yamamoto H,
Yamanaka S, Nakahata T, Heike T.
Generation of skeletal muscle stem/progenitor cells from murine induced
FASEB J. 24(7)2245-2253, 2010.
2. Chang H, Yoshimoto M, Umeda K, Iwasa T, Mizuno Y, Fukada SI, Yamamoto H,
Motohashi N, Suzuki YM, Takeda S, Heike T, Nakahata T.
Generation of transplantable, functional satellite-like cells from mouse embryonic
stem cells.
FASEB J. 23(6) 1907-1919, 2009.
3. Yokoo N, Baba S, Kaichi S, Niwa A, Mima T, Doi H, Yamanaka S, Nakahata T, Heike T.
The effects of cardioactive drugs on cardiomyocytes derived from human induced
Biochem Biophys Res Com. 387(3) 2482-2488, 2009.
4. Niwa A, Umeda K, Chang H, Saito M, Okita K, Takahashi K, Nakagawa M.,
Yamanaka S, Nakahata T, Heike T.
Orderly Hematopoietic Development of Induced Pluripotent Stem Cells via Flk-1+
Hemoangiogenic Progenitors.
J. Cell. Physiol. 221(2):367-377, 2009.
5. Higashi AY, Ikawa T, Muramatsu M, Economides AN, Niwa A, Okuda T, Murphy AJ,
Rojas J, Heike T, Nakahata T, Kawamoto H, Kita T, Yanagita M.
Direct hematological toxicity and illegitimate chromosomal recombination caused
by the systemic activation of CreERT2.
J Immunol. 182(9):5633-40, 2009.
6. Kato M, Sanada M, Kato I, Sato Y, Takita J, Takeuchi K, Niwa A, Chen Y, Nakazaki
K, Nomoto J, Asakura Y, Muto S, Tamura A, Iio M, Akatsuka Y, Hayashi Y, Mori H,
Igarashi T, Kurokawa M, Chiba S, Mori S, Ishikawa Y, Okamoto K, Tobinai K,
Nakagama H, Nakahata T, Yoshino T, Kobayashi Y, Ogawa S
Frequent inactivation of A20 in B-cell lymphomas.
Nature 459:712-716,2009.
35
Haruhisa Inoue
Profile
M.D., Ph.D.
Associate Professor
Members
●
Associate Professor
Haruhisa Inoue
●
Researchers
Shiho Kitaoka
Naoki Yahata
Naohiro Egawa
●
Technical Staff
Kayoko Tsukita
Mitsuyo Kawada
Yoshiko Karatsu
Yumiko Iwamoto
Fumihiko Adachi
●
Research Assistant
Takayuki Kondo
(Ph.D. Student, Kyoto University
Faculty of Medicine Dept. of
Neurology )
●
Secretary
Kazumi Murai
36
Establishment of
neurodegenerative disease-specific
iPS cells, induction of their
differentiation and analysis
It is one of the urgent topics in the
society of aging population to develop
methods of treatment for intractable
neurodegenerative diseases induced by
degeneration or loss of cells in the central
nervous system (CNS) such as amyotrophic
lateral sclerosis (ALS), Parkinson s disease
and Alzheimer s disease (Protein Folding
and Misfolding: Neurodegenerative Diseases,
Focus on Structural Biology series vol. 7,
Springer, New York, p97-110). Following
recent advances in molecular biological
studies, understanding of the molecular
mechanisms for these diseases has been
deepened. However, no method for radical
prevention or treatment of such diseases
has yet been established. The primary locus
of lesion in patients with ALS is reported to
be nerve cells called motor neurons which
control the voluntary movements of the
living body. Because motor neurons are
located in the CNS enclosed by the skull or
spinal vertebrae, there was a limitation of
direct analysis of the features of these
diseases. For this reason, studies of these
diseases have conventionally focused on
genetic analysis and biochemical and
histological analyses of histopathologically/
genetically modified animals or cell models.
Born in Kyoto City in 1967. Graduated from
Acquired PhD in Medicine at Kyoto University
Graduate School. After services at the
Department of Neurology, Kyoto University
Hospital, and the Department of Neurology,
Sumitomo Hospital, Studied for 2 years from
1997 at the National Institute of Neuroscience,
and the Department of Neuropathology,
University Medical School of Pécs, Hungary.
Then, after working at the RIKEN Brain Science
Institute and the Harvard Medical School
McLean Hospital, joined the Department of
Clinical Neurology, Kyoto University Graduate
School of Medicine in 2005. iPS Cell Research
Center, Institute for Integrated Cell-Material
Science, Kyoto University in 2009 before taking
the current position.
In other word, past studies pertained only
to indirect analysis of the features of these
diseases from which not a few patients
suffered. Our laboratory is now aimed at
controlling intractable neurodegenerative
diseases such as ALS on the basis of further
deepened understanding of their molecular
mechanisms enabled by the use of patientderived cells (cells which were totally
impossible to obtain in the past but are
now possible to prepare thanks to the
discovery of iPS cell technology).
Neurodegenerative disease-specific iPS
cells are considered to be applicable in
three directions: disease modeling, disease
materials and transplant therapy (Exp Cell
Res. 316; 2560-4, 2010). Disease modeling
pertains to recapitulate the diseasephenotype in the culture dish, followed by
exploration of the pathological mechanism
and generation of the possibility to clarify
the features of not only familial diseases but
also sporadic diseases. Furthermore, it is
also applicable as the platform for drug
discovery screening and a new-generation
research tool for neurodegeneration
imaging. If disease materials which are
usually difficult to obtain can be used for
molecular biological analysis, there is a
potential of revealing previously unknown
pathomechanistic factors. It is also possible
to create nerve cells and glial cells used for
transplant therapy.
Challenging to elucidate the intractable diseases of the central
nervous system
iPS cells derived from a patient with amyotrophic
lateral sclerosis (ALS)
During the current year, our laboratory
conducted research, taking further steps
towards systematical establishment of
neurodegenerative disease-specific diseases
(ALS, etc.), inducing differentiation into
cells of the nerve systems, and reproducing
the microenvironment (niche) of
neurogenerative diseases in vitro.
In the studies conducted to date, we
found that the efficiency of inducing
differentiation of disease-specific iPS cells
into disease-targeted cells varies among iPS
cell lines even when the cells were derived
from the same individual. Following this
finding, we developed a technique by which
differentiation into neurons (disease-
A research topic at the Inoue Laboratory is control of intractable neurodegenerative
diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer's disease on which
no radical method for prevention or treatment is available. iPS cells are established from
the cells associated with the diseases, followed by induction of their differentiation into
cells of the central nervous system (motor neurons, etc.) and detailed analysis of this
process, with a goal of elucidating the essential features of the diseases and exploring
methods of their prevention and treatment.
The research during the current year set a goal at preparing iPS cells from the skin
cells of patients with ALS, etc. and to induce their differentiation into motor neurons to
create a condition similar to the disease concerned. In addition, the research during the
current year was designed to establish disease-specific iPS cells in an efficient and
systematical manner and to induce these cells into nerve cells. Through these attempts,
molecules associated with the diseases and having the potential of playing a key role in
responses of the diseases to treatment are now being identified.
targeted cells) can be rapidly induced,
yielding neurons in a pure form. Using this
technique, we have conducted thorough
analyses, including transcriptome analysis
and are unveiling the previously unknown
molecular features of diseases and new
targets for drug creation. Our research in
this direction is expected to clearly
illustrate the relationship between protein
misfolding and neurodegeneration in the
cells from humans, although this
relationship was conventionally analyzed
only indirectly. Furthermore, we have
prepared the platform for selection of
multiple existing drugs as candidates for
valid means of treating familial ALS due to
mutant SOD1（J Biomol Screen. in press）.
The responses to the thus identified
candidate drugs are now being evaluated
using iPS cell-based disease reproduction
models and animal models.
Findings from analysis of diseases
using iPS cell technology have been
increasingly accumulated. Although
disease modeling has conventionally
been confined to some particular diseases
whose onset involves genetic factors very
closely, it may be applied more widely
from now, including neurodegenerative
diseases as well, if studies on aging
factors, and environmental factors are
advanced.
Publications
1. Murakami G, Inoue H, Tsukita K, Asai
Y, Amagai Y, Aiba K, Shimogawa H,
Uesugi M, Nakatsuji N, Takahashi R.
Chemical library screening identifies
a small molecule that downregulates
SOD1 transcription for drugs to treat
ALS.
J Biomol Screen. in press.
2. Inoue H.
Neurodegenerative disease-specific
induced pluripotent stem cell
research.
Exp Cell Res. 316(16);2560-2564, 2010.
Inclusion body within motor neurons of a patient with ALS (Courtesy: Dr. Hidefumi Itoh, Department of Clinical
Neurology, Kyoto University Graduate School of Medicine)
3. Inoue H, Kondo T, Lin L, Mi S, Isacson
O, Takahashi R.
Protein misfolding and axonal
protection in neurodegenerative
diseases., In Protein Folding and
Misfolding: Neurodegenerative
Diseases, Focus on Structural Biology
series vol. 7, Springer, New York,
97-110, 2009.
37
Hidetoshi Sakurai
Profile
M.D., Ph.D.
Lecturer
Born in Ikeda Town, Gifu Prefecture in 1973.
Graduated from Nagoya University School of
Medicine in 1998. Trainee and then services as a
nephrologist at the Nagoya Ekisaikai Hospital.
Studied at the doctoral course of Nagoya
University Graduate School of Medicine in 2001
through 2005. Acquired PhD in Medicine. During
this period, researcher at the Stem Cell Group
(Nishikawa Laboratory) of the RIKEN Kobe
Institute Center for Developmental Biology.
Since 2005, Department of Immunology, Nagoya
University Graduate School of Medicine as a
Research Resident of the Japan Foundation for
Aging and Health. Since June 2008, Specific
Researcher at the iPS Cell Research Center,
Institute for Integrated Cell-Material Science,
Kyoto University. Current position since
November 2009.
Mature skeletal muscle differentiated from satellite
cells of mouse ES cell origin
Members
●
Lecturer
Hidetoshi Sakurai
●
Technical Staff
Izumi Maki
Tokiko Nishino
Junichi Kondo
●
Graduate Students
Hiroshi Sakai
Akihito Tanaka
Emi Shoji
●
Secretary
Katsura Noda
Our laboratory is aimed at establishing
methods for treatment of intractable
muscular diseases, particularly muscular
dystrophy. We have set our goal at two
processes of treatment. One is cell
transplant therapy. The other is
development of drugs. The former utilizes
the precursor cells (differentiated from iPS
cells) as a source of transplant. Its efficacy is
now under evaluation in experiments using
animal models. The latter uses iPS cells
established from patients, aimed at
developing disease model in vitro and
utilizing them as a tool of drug
development.
1．Cell transplant therapy
Duchenne muscular dystrophy
(DMD), a severe and the most frequent
type of muscular dystrophy, is a potentially
fatal muscular disease transmitted via the
dominant X-linked trait. Regarding the
etiology of this disease, it is known that
deficiency of the protein dystrophin causes
fragility of muscular cell membrane,
leading to induction of chronic
inflammation and marked muscular
atrophy. As a strategy for cell transplant
therapy for DMD, we set a goal at achieving
differentiation of the transplanted cells into
satellite cells (musculoskeletal stem cells)
and subsequent survival of the cells in the
recipient. We expect that the satellite cells
38
derived from the transplanted cells will
proliferate within the recipient and
contribute to regeneration, resulting in
increase in normal myofibers expressing
dystrophin and curing of the disease.
Previous studies using mouse ES cells
demonstrated that platelet-derived growth
factor receptor α(PDGFR-α) can be used
as a marker of paraxial mesoderm
(precursor tissue for bone, cartilage and
skeletal muscle) (Reference 3). It has been
reported that transplantation of PDGFR-α
positive cells into a mouse model of
muscular injury resulted in differentiation
into satellite cells (skeletal muscle stem
cells) (Reference 2). It has also been shown
that when differentiation of ES cells was
induced in a serum-free culture system
supplemented with factors known to play
an important role in paraxial mesoderm
formation during early mouse
development, differentiation into PDGFRα positive paraxial mesodermic cells took
place with high efficiency (Reference 1).
Although the extent of growth factor
demand differed between mouse iPS cells
and ES cells. Induction of PDGFR-α
positive cells was possible from mouse iPS
cells at an efficiency comparable to that
with ES cells. When mouse iPS cells
permanently expressing DsRed (red
fluorescence-emitting protein) were
induced to differentiate, followed by
isolation of PDGFR-α positive cells and
their transplantation into the skeletal
mouse of a mouse model of DMD,
differentiation into satellite cells was noted
in some cells. Furthermore, dystrophin
positive, DsRed positive myofiber
formation was noted, thus indicating
contribution of these cells to muscular
degeneration. However, since the survival
rate of these cells in the recipient was very
low, we are now exploring a method for
transplantation with better survival.
Resarch is under way also on induction
of skeletal muscle precursor cells from
human iPS cells. We tested several methods
previously reported for human ES cells, but
these methods often failed to yield
reproducible results. The lack of
reproducibility seemed to be attributable to
the inability to identify the stage at which
differentiation failed because induction was
not proceeded in a step-wise manner. For
this reason, we are attempting a method by
which paraxial mesoderm is initially
induced, followed by step-wise induction of
differentiation into the somite and then to
the dermomyotome. To date, we have
succeeding in isolating a group of cells at
the somite level. Now, we are analyzing
whether or not these cells can differentiate
into dermomyotome, eventually becoming
satellite cells.
2．Development of a disease
model in vitro
To reproduce muscular dystrophy in
vitro and to use this technique as a tool for
drug development, it is essential that
differentiation into mature skeletal muscle
can be induced at a very high efficiency.
One strategy for this end involves
purification with the use of a marker more
specific to skeletal muscle than is PDGFRα. At present, we are preparing human iPS
cells with a GFP knock-in transcription
factor (serving as a marker). Another
strategy involves induction of
differentiation through forced expression
of transcription factor.
We plan to apply this technique first to
DMD, a severe disease with a large number
of patients. Jointly with the Nakahata
Laboratory of our center, we plan to use iPS
cells prepared from fibroblasts of DMD
patient origin. We anticipate that the point
of action by the drugs created with this
technique is the process in which
inflammation becomes chronic. We will
attempt to develop a system enable
reproduction and visualization of chronic
Establishment of iPS cells from patients with muscular dystrophy
and their application to cell therapy and drug development
No valid means of treatment has been established for muscular dystrophy, an intractable
disease involving chronic inflammation and atrophy of skeletal muscles. The Sakurai
Laboratory is attempting to establish cell therapy for this disease, through inducing
differentiation of iPS cells into skeletal muscle stem cells and subsequently transplanting
these cells to increase skeletal muscle in the patients.
In the studies conducted hitherto using mouse ES cells, differentiation into skeletal
muscle stem cells within the skeletal muscle of mice with muscular injury was successfully
induced with the use of cells carrying platelet-derived growth factor receptor α (PDGFRα). Also with mouse iPS cells, induction of PDGFR- α positive cells and differentiation into
skeletal muscle stem cells in a mouse model of DMD have been reported. Increase in
myofibers has also been shown, but the Sakurai lab. is exploring an optimal method of
transplantation to achieve higher efficiency of cell survival in the recipient. Furthermore,
studies on methods for step-wise induction of differentiation from human iPS cells are
also under way.
In addition, a study for development of a system enabling visualization of intracellular
molecules has started, with a goal of applying to the so far developed mouse and human
iPS cells and patient-derived disease-specific iPS cells to clarification of the features of
muscular dystrophy and development of drugs for treatment of this disease.
inflammation. Furthermore, jointly with
the Department of Neurology, Kumamoto
University, we will analyze iPS cells derived
from patients with Miyoshi type muscular
dystrophy arising from dysferlin deficiency.
With this type of dystrophy, distal muscles
are primarily involved in pathogenesis.
Regarding the mechanism for onset of this
type of dystrophy, delayed regeneration of
muscular cell membrane due to dysferlin
deficiency has been considered to be
responsible for the disease. Because studies
on this type of dystrophy have not been
advanced as compared to studies on DMD,
our goal for the time point is to develop a
tool which can contribute to clarification of
this disease.
Publications
1. Sakurai H, Inami Y, Tamamura Y,
Yoshikai T, Sehara-Fujisawa A, Isobe K.
Bidirectional induction toward
paraxial mesodermal derivatives
from mouse ES cells in chemically
defined medium.
Stem Cell Res. 3(2-3):157-69, 2009.
2. Sakurai H, Okawa Y, Inami Y, Nishio
N, Isobe K.
Paraxial mesodermal progenitors
derived from mouse embryonic stem
cells contribute to muscle
regeneration via differentiation into
muscle satellite cells.
Stem Cells 26(7) :1865-73, 2008.
3. Sakurai H, Era T, Lakt LM, Okada M,
Nakai S, Nishikawa S, Nishikawa SI.
In vitro modeling of paraxial and
lateral mesoderm differentiation
reveals early reversibility.
Stem Cells 24(3): 575-86, 2006.
4. Tada S, Era T, Furusawa C, Sakurai H,
Nishikawa S, Kinoshita M, Chiba T,
Nishikawa SI.
Characterization of mesendoderm: a
diverging point of the definitive
endoderm and mesoderm in
embryonic stem cell differentiation
culture.
Development 132(19): 4363-74, 2005.
Mature skeletal muscle differentiated from mouse
iPS cells in vitro
39
Department of Regulatory Science
Takafumi Kimura
Profile
M.D., Ph.D.
Head of FiT & Professor
Born in Sakai City, Osaka Prefecture in 1961.
Graduated from Nara Medical University.
Acquired PhD in Medicine. After clinical
experience (internal medicine) at the Third
Department of Internal Medicine of Osaka
University Hospital and its affiliated hospitals,
began research on human hematopoietic stem
cell proliferation and differentiation at the
Department of Hygiene, Kyoto Prefectural
University of Medicine. After working at the
Department of Hygiene, Kansai Medical
University and the Research Division of Japanese
Red Cross Osaka Blood Center, assigned to the
current position in April 2010.
Fig.1 Open cell culture laboratories
Members
●
Professor
Takafumi Kimura
●
Technical Staff
Miho Tani
●
Secretary
Soyoko Kadoya
In April 2010 when the Center for iPS Cell
Research and Application (CiRA) was
founded, we began research towards the
goal of preparing iPS cells at the Facility for
iPS Cell Therapy (FIT) organized within
the CiRA. The mission of FiT is preparing
safe and high-quality cells indispensable
for realization of regenerative medicine
(cell therapy) with the use of iPS cells. To
facilitate achieving this mission, it is
essential to arrange two major elements of
FiT, i.e., facility/equipment and system.
Facility/equipment validation and
performance evaluation
Validation and performance evaluation
were carried out, covering the air
conditioning and security systems and all
Fig.2 Closed cell culture laboratories installed with isolators
40
equipments and devices installed in the 4
conventional type open cell culture
laboratories (Fig. 1) and the 2 closed cell
culture laboratories installed with isolators
(Fig. 2). The capability of preparing cells in
an aseptic and safe manner was thus
confirmed.
Organizational establishment
A system indispensable for FiT
management pertains to organization and
documentation. First we attempted to
establish an organization by formulating
internal rules and founding the Steering
Committee. These measures are expected to
ensure transparent FiT management and
third party evaluation. Production Division
and Quality Control Division were
organized under the FiT Director as units
responsible for jobs complying with GMP
(Good Manufacturing Practice: rules on
production and quality control).
Production Administrator and Quality
Administrator were appointed at these
units, respectively. The Steering Committee
checks and discusses the research topics,
budgets, documentation systems including
SOP (Standard Operating Procedures), and
status of job implementation based on such
documents. Before research on a given
topic is started, it needs to be approved by
the CiRA Director after discussion at the
Management Committee. More practical
issues, pertaining to culture methods, and
quality test methods, are discussed at the
working level conference, a panel organized
under the Steering Committee.
Development of
documentation system
At the Kyoto University Hospital, many
attempts of cell therapy have been made,
including pancreatic head cell
transplantation, dendritic cell
transplantation, and bone marrow (BM)derived mesenchymal stem cell (MSC)
transplantation for aseptic bone necrosis.
With close cooperation of the staff
members of the Center for Cell and
Molecular Therapy (head: Taira Maekawa)
experienced with preparation of cells used
for these clinical studies, we began to
formulate documents, beginning with those
requiring high levels of priority, i.e., 3 sets
of control standards required under GMP
(production control standards, quality
control standards and hygienic control
standards) as well as the validation
procedure manual, the document control
manual, and the self-inspection manual. At
present, proofreading is under way towards
completion of FiT Documentation System
Version 1 composed of 60-odd sets of
standards, and we are now preparing for
the start of experiments on trial operation
on these documents.
Trial operation
For the purpose of preparing human
iPS cells (and iPS cell-derived tissue cells)
and their clinical application, we are
forming a new framework on methods of
culture and quality assessment to prepare
safe and high quality cells. In parallel to
such efforts, FiT plans to begin trial
operation on the methods of culture for
human marrow cells (including
mesenchymal stem cells). Trial
Preparation of safe and high quality iPS cells playing a key role
in realization of regenerative medicine
If iPS cells are assumed to be used during clinical practice, they need to be assured as to
safety, as is required of medicines in general. Production and stable supply, complying
with the GMP, are essential for iPS cells used for such purposes. It is also essential that
iPS cells can be delivered to medical facilities in the form of viable cells while keeping
them fresh. Prof. Takafumi Kimura, who directs the cell preparation facility (FiT: Facility
for IPS Cell Therapy) recently founded in the new building, will be engaged in preparing
safe and high quality iPS cells which are indispensable for realization of cell therapy with
iPS cells.
To date, installment of devices in the FiT cell culture laboratory, their adjustment
and evaluation of their function have been implemented, accompanied by confirmation
of the air-conditioning and security systems and formulation of the organization and
rules for operation of the laboratory.
Numerous documents and procedure manuals needed for production and
utilization processes are now being prepared. In the near future, an experiment on trial
operation of this laboratory will be undertaken, covering a series of processes, including
material cell collection at the Kyoto University Hospital, transport of collected cells,
manipulation of BM-derived MSCs at FiT and delivery of the cultured MSCs to the
hospital. Now, the steps preparing for such an experiment are being taken.
implementation of a series of steps
(collection of bone marrow at the Kyoto
University Hospital, transport of the
collected cells, receipt at FiT, cell isolation,
culture, frozen storage, thawing, re-culture
and delivery to the hospital) will enable
repeated review of the SOP and processes.
Validation and verification are
indispensable not only on the culture
methods but also on many devices and
testing methods employed for quality
assessment and frozen storage. We will
satisfy these requirements soon and begin
culture of marrow cells.
Publications
1. Yagita M, Yasui K, Hori Y and Kimura T.
Reversible IgA deficiency after severe Gram-negative bacteria infection in a case
with systemic sclerosis.
Modern Rheumatol. 20, 2010. in press.
2. Yasui K, Angata T, Matsuyama N, Furuta RA, Kimura T, Okazaki H, Tani Y, Nakano
S, Narimatsu H and Hirayama F.
Detection of anti–Siglec-14 alloantibodies in blood components implicated in
nonhemolytic transfusion reactions.
Transfusion in press.
3. Kimura T, Matsuoka Y, Murakami M, Kimura T, Takahashi M, Nakamoto T, Yasuda K,
Matsui K, Kobayashi K, Imai S, Asano H, Nakatsuka R, Uemura Y, Sasaki Y, and Sonoda Y.
In vivo dynamics of human cord blood-derived CD34- SCID-repopulating cells
using intra-bone marrow injection.
Leukemia 24:162-168, 2010.
4. Matsuyama N, Hirayama F, Wakamoto S, Yasui K, Furuta RA, Kimura T, Taniue A,
Fukumori Y, Fujihara M, Azuma H, Ikeda H, Tani Y, Shibata H.
Application of the basophil activation test in the analysis of allergic transfusion
reactions.
Transf Med. 19:274-277, 2009.
5. Matsuyama N, Hirayama F, Yasui K, Kojima Y, Furuta RA, Kimura T, Taniue A,
Fukumori Y, Tani Y, Shibata H.
Non-HLA white cell antibodies in nonhemolytic transfusion reactions.
Transfusion 48:1526-1528, 2008.
6. Yasui K, Furuta RA, Matsuyama N, Fukumori Y, Kimura T, Tani Y, Shibata H,
Hirayama F.
Possible involvement of heparin-binding protein in transfusion-related acute lung injury.
Transfusion 48:978-987, 2008.
41
Takashi Aoi
Profile
M.D., Ph.D.
Professor
Born in Kobe City in 1973. Graduated from Kobe
University School of Medicine. Completed the
doctoral course at Kyoto University Graduate
School of Medicine. Acquired PhD in Medicine.
After graduation from medical school, engaged
in clinical practice and research as an
gastroenterologist. Began basic research on iPS
cells at the Shinya Yamanaka Laboratory in 2005.
Since 2009, engaged in dealing with regulations,
etc, aiming to realize clinical application of iPS
cells as soon as possible.
Induction of human iPS cell differentiation into liver
cells
Immunostaining for a marker of differentiation into
liver (AFP). The nuclei stained blue with Hoechst.
Members
●
Professor
Takashi Aoi
●
Graduate Students
Masatoshi Kajiwara
Akiko Fukuhara
Yuji Mochiduki
Nobu Oshima
●
Technical Staff
Tokiko Ohkame
Yukari Matsukawa
Tomomi Ito
●
Secretary
Yumi Higuchi
1. Dealing with regulations
towards clinical application of
iPS cells
1) Adjustment of regulations to match the
current status of science
It is hoped that cell transplantation
therapy with iPS cells will be realized in the
near future. Now, regulations are needed to
govern such therapy appropriately. Because
iPS cells have various features not found in
existing materials for healthcare, there are
many aspects in which existing regulations
cannot be directly applied. Under such
circumstances, arrangement of guidelines,
etc. pertaining to cell therapy with the use
of iPS cells are now being made at the
initiative of the Ministry of Health, Labour
and Welfare (MHLW). Cell therapy with
iPS cells can be attempted under two tracks:
(1) clinical study carried out pursuant to
the Medical Practitioner s Act, and (2)
clinical trial carried out pursuant to the
Pharmaceutical Affairs Act. Our research
group has been participating in both of the
Working Group of the Expert Committee
on Reviewing the Guidelines for Clinical
Studies with Human Stem Cells (related to
Track 1) and the Conference of the Fiscal
2010 Study Group (Hayakawa Group) on
Ensuring the Quality and Safety of Cell/
Tissue-Processed Pharmaceutical Products
Derived from Human Stem Cells (related
to Track 2). Thus, our group has been
involved in development of appropriate
Karyotype observed with conventional Giemsa staining / Karyotype observed with G-band method
Tests conducted under a setting optimal for human iPS cells/ES
42
Involvement in Development of Regulations Important for
Clinical Application and in Evaluation of iPS cells
Multicolor FISH (mFISH） At CiRA, mFISH is carried out
as needed if abnormalities are revealed by observation
with conventional Giemsa staining or G-band method.
regulations through reporting our view on
the current status of science and
perspectives for the future.
2) Adjustment of research to match the
regulations
The existing regulations and the
underlying views are partially applicable to
regenerative medicine with the use of iPS
cells. On these parts of the existing
regulations and views, it is desirable to
adjust the research to match them. Within
the CiRA, several working groups have
been organized to facilitate research beyond
the frameworks of study groups with a goal
of clinical application of iPS cells. We have
been participating in these working groups
and proposing goals and their
implementation plans from the viewpoint
of matching the iPS cell technology to
regulations.
2. Evaluation of genomic stability
In establishing and maintaining iPS
cells, genomic stability is a matter of high
concern. Our group has been approaching
this issue with two methods.
1) Evaluation of genomic stability using
high resolution SNP arrays
This research is under way primarily by
Akiko Fukuhara, jointly with Specially
Appointed Associate Professor Seiji Ogawa
(University of Tokyo). This research is
designed to analyze chromosomal changes
(changes in the number of copies) with the
use of high resolution SNP arrays in human
iPS cells established with various
technologies, i.e., established from different
cells, with different methods of factor
introduction, and by introduction of
different factors. At the same time, the
research is designed to evaluate the
influence of passaging on chromosomal
changes using samples of the same clone
passaged for varying numbers of
generation.
Because iPS cells are a new concept of materials used for healthcare, arrangement of
new regulations while making adjustment with existing regulations is now under way to
facilitate the start of cell therapy with the use of iPS cells.
Prof. Takashi Aoi is now involved in development of regulations for such therapy as
a member of both the Working Group of the Expert Committee on Reviewing the
Guidelines for Clinical Studies with Human Stem Cells and the Conference of the Fiscal
2010 Study Group (Hayakawa Group) on Ensuring the Quality and Safety of Cell/
Tissue-Processed Pharmaceutical Products Derived from Human Stem Cells" organized
at the initiative of the MHLW.
In addition, in working groups organized within CiRA, the Aoi group is expected to
make proposals and accept consultation related to adjustment and utilization of iPS cells
to satisfy both old and new regulations and to evaluate the practically established iPS
cells through analysis of their chromosomal changes and karyotype.
At the Aoi Laboratory, research is under way on induction of human iPS cell
differentiation into liver cells, including investigation of differences in the manner of
manifestation of the characteristics of iPS cell differentiation through analysis of gene
expression and epigenome in undifferentiated cells and cells during the course of
differentiation.
A new technique of data analysis has
also been introduced, enabling in-house
analysis of data. In this way, it is now
possible to compare data obtained at our
laboratory with the raw data previously
collected at other research institutes and
accessible on the web.
2) Human iPS/ES cell karyotype analysis
The karyotype analysis laboratory has
been set up and put into operation by
Tokiko Ohkame and Yukari Matsukawa.
Optimum setting for karyotype analysis of
human iPS/ES cells has been established at
this laboratory. In addition to the routine
techniques (conventional Giemsa staining
and G band method), mFISH method and
Q band method are also applicable.
Furthermore, a device for automated
detection of cell division metaphase has
been installed and put into operation,
enabling rapid and efficient karyotype
analysis. This laboratory now conducts the
above-mentioned analysis assigned from
researchers across our center and provides
consultation about the test data.
3. Induction of human iPS cell
differentiation into liver cells
At the initiative of Masatoshi
Kajiwara, research is now under way on
induction of human iPS cell
differentiation into liver cells. The
recently developed method for induction
of differentiation has been shown to be
capable of preparing cells resembling
liver cells in terms of morphology, gene
expression and function in a stable and
reproducible manner. Studies using this
method have revealed that the
characteristic of differentiation into liver
cells varies among multiple iPS cell lines
even when these cell lines were
established by the identical method from
the cells of the same donor. To elucidate
the molecular mechanism for such
variances, we are now analyzing gene
expression, epigenome, etc. in
undifferentiated cells and cells during the
course of differentiation.
Publications
1. Imamura M, Aoi T, Tokumasu A, Mise N, Abe K, Yamanaka S, Noce T.
Induction of primordial germ cells from mouse induced pluripotent stem cells
derived from adult hepatocytes.
Mol Reprod Dev. 77(9):802-811, 2010.
M, Tanabe K, Ohnuki M, Ogawa D, Ikeda E, Okano H, Yamanaka S.,
Nat Biotechnol. 27(8):743-745, 2009.
3. Aoi T, Yae K, Nakagawa M, Ichisaka T, Okita K, Takahashi K, Chiba T, Yamanaka S.
Generation of pluripotent stem cells from adult mouse liver and stomach cells.
Science 321(5889):699-702, 2008.
43
Isao Asaka
Profile
M.D., Ph.D.
Associate Professor
Born in 1959, in Ohta-ku, Tokyo. Graduated from
Kitasato University School of Pharmacy.
Graduated from the master s course at the
Kitasato University Graduate School of
Pharmaceutical Sciences. Received PhD in
Pharmacology in 1994. Lecturer at Jikei Group
Tokyo College of Medico Pharmaco Technology
from 1986-1989. AGC Techno Glass Co., Ltd. from
1989 - 2008. During this period, Research
Associate at the Kitasato University School of
Pharmacy, Visiting Researcher at the Institute of
Medical Science, University of Tokyo, and so on.
Japan Science and Technology Agency(JST)
Yamanaka iPS Cell Project Researcher,
concurrently assigned to Lecturer at the Center
for iPS Cell Research and Application(CiRA),
Institute for Integrated Cell-Material Science,
Kyoto University in October 2008. Associate
Professor at the CiRA, Kyoto University (current
position), since April 2010.
System for storing disease-specific iPS cells
Members
●
Associate Professor
Isao Asaka
●
Researcher
Koichi Igura
●
Technical Staff
Ayako Nagahashi
Toshie Kusunoki
Aya Kishida
Sayori Yano
Monica Murakami
●
Secretary
Katsura Noda
44
Activity for dissemination of
iPS cell technology
As the framework of the actions to
promote the dissemination of iPS cell
technology, we have held technical lectures
focused on human iPS cell establishment
and maintenance culture technologies
(twice a year) and practical trainings
(about 4 times a year) to researchers
outside the Center for iPS Cell Research
and Application (CiRA). At each session of
these instructional programs, we execute
questionnaires to confirm the level of
understanding amongst participants. In
addition, the status of iPS cell technology
application in the participant s home
institution is investigated with
questionnaires. On the basis of the results
from these investigations, we plan followup programs as needed. During the current
year, the first lecture course and the second
practical training course were provided in
English to facilitate dissemination of the
CiRA s iPS technology as a useful standard
method in drug discovery and disease
research both in Japan and overseas. The
first lecture course was provided with the
cooperation of PIs (laboratory chiefs),
including Lecturer Masato Nakagawa and
Lecturer Kazutoshi Takahashi from the
and Associate Professor Kenji Osafune from
the Department of Cell Growth and
Differentiation. The lectures about current
human iPS cell technologies were explained
under the titles of Generation, culturing
and maintenance of human iPS
cells (Asaka), Development and recent
trend of generation of human iPS
cells (Lecturer Nakagawa), Evaluation of
human iPS cells (Lecturer Takahashi) and
Generation and analysis of disease specific
iPS cells (Associate Professor Osafune).
There were 38 participants in this program,
including 15 foreign researchers, of whom
more than 70% were staying in Japanese
universities as students or researchers.
According to the result of questionnaire
survey about the remark of this lecture
course, 85% of the participants indicated to
be Excellent or Good". The follow-up
questionnaires were executed to the
participants in the practical training in
2009, revealed a frequent request on lecture
as to the methods for quality check of
prepared iPS cells and for induction of
differentiation. In accordance with these
results, we plan to provide the second
lecture course covering these requests
within the current year.
Regarding the practical training, the
training room for iPS cell establishment
and maintenance culture had been
constructed in the CiRA building. We
advanced the preparation to start the
practical training for human iPS cell
establishment and maintenance culture
within our facility, and had held the first
practical training program at the end of
June in this year. To date, a total of 24
attendees participated in the training about
establishment of human iPS cells by the
retrovirus method and practical steps of
cell maintenance culture. According to the
result of questionnaire survey about the
remark of these training courses, 67% of
the participants indicated Excellent and
33% were Good with the overall training.
A follow-up questionnaire survey was
executed to the participants in the practical
training held in 2009 to investigate the
status of iPS cell technology application in
the participant s home institutions.
According to the result of follow-up survey,
40% were utilizing human iPS cells for
their research. Among those who answered
to be utilizing human iPS cells for their
research, 75% were using the human iPS
cells established by themselves, thus
indicating that the practical training was
effective at least to some extent. Of the
remaining 60% respondents, 40% were
preparing to start human iPS cell research
and 20% had given up utilization. The
reasons listed for giving up utilization were
budget restrictions and changes in project
situations, and were not related to the
design of the training program provided to
them.
Establishment of
disease-specific iPS cells
We initially induce fibroblasts from the
skin biopsies collected from various
patients supplied from the Graduate School
Providing lectures and practical training to disseminate iPS cell
establishment technology and the methods for iPS cell handling
The Asaka Laboratory is in charge of disseminating the human iPS cell establishment
and maintenance culture technology and quality checking methods, which are
indispensable for basic research and clinical application of iPS cells. During the current
year, 2 sessions of lecture and 4 sessions of practical training were provided periodically.
These programs primarily covered researchers outside the CiRA. When the level of
understanding by the participants and the extent of application of the learned
technology were investigated with questionnaire surveys, high degrees of satisfaction
with the programs were revealed. Moreover, the group will plan follow-up programs
and further advanced lecture and training programs.
Establishment and selection of disease-specific iPS cells derived from specimens of
various patients have also been carried out, and the iPS cells have been supplied to
relevant units within the CiRA and outside research institutes. During the current year,
fibroblast lines were established from the skin-biopsies of more than 30 patients by
December, and disease-specific iPS cells were established from those fibroblasts of
about 10 patients. The group are also exploring a method for more efficient
establishment of disease-specific iPS cells.
of Medicine and other PIs of CiRA, and
store 10 or more cryo-vials contained
frozen fibroblasts. Disease-specific iPS cells
will be established from the thus ensured
stock of patient-derived fibroblasts and will
be provided as materials for researching
pathogenesis and the development of new
drugs within CiRA and external
laboratories. For continuity and reference,
the original somatic cell samples will be
stored and managed along with established
disease-specific iPS cells. Furthermore, in
cooperation with the Department of
Reprogramming Science and the
Department of Clinical Application, we try
to establish disease specific iPS cells from
other somatic cell materials (ex. blood cells,
etc.) except fibroblasts, to find alternative
somatic cell materials as a safer source of
disease-specific iPS cells. Since the new
CiRA building was completed in the early
part of current year, we had set up the
new dedicated culture room for
establishment of disease-specific iPS cells.
We had also constructed a system for
preparation of fibroblast lines from
patient skin-biopsy and preparation of
disease-specific iPS cell lines from those
fibroblast lines in a systematical manner.
By December of the current year, skinbiopsies were collected from more than
30 patients, to induce fibroblasts and to
store them frozen. Using the fibroblasts
derived from about 10 patients, diseasespecific iPS cells have been established. To
improve the efficiency of establishment
of disease-specific iPS cells, we had
studied titer checking methods for
lentivirus and retrovirus vector solutions
for use in introduction of
reprogramming factors. We found that
titer checking is possible to some extent
by p24-ELISA for lentivirus vectors and
by q-PCR for retrovirus vectors.
Publications
Practical training
1. Asaka I. Adipocytes differentiation
culture from mesenchymal stem cells
- Time lapse analysis of
differentiation process- (In Japanese)
Huh N. and Nakamura Y. ed.,
Experiment Handbook for Cultured
Cells, Yodosha Co., Ltd., pp125-130,
2008.
2. Asaka I. Standard Technique of Cell
Culture (In Japanese),The Japanese
Tissue Culture Association ed., The
Japanese Tissue Culture Association,
10-23, 2007.
3. Asaka I. Cynomolgus monkey ES
cells. (In Japanese) Saibo 36(2) 22-23,
2004.
45
Research Projects
iPS Cell Research Project for Regenerative Medicine
The iPS Cell Project for Regenerative Medicine, led by Shinya
Yamanaka, is one of the 30 projects of the Funding Program for
World-Leading Innovative R&D on Science and Technology
(FIRST Program), created by the Cabinet Office of Japan to push
Japan's most advanced research projects. Its objective is to
compare properties of iPS cell lines derived from different sources
and using different generation methods and realize the global
standardization of iPS cell technology as early as possible.
Web site：http://www.cira.kyoto-u.ac.jp/ips-rm/?lang=en
The Project for Accelerating Clinical Application of iPS cells
CiRA is part of a "Super Tokku" project, backed by the Cabinet
Office of Japan, in which the participating organizations are Kyoto
University, Keio University, the University of Tokyo, RIKEN,
Astellas Pharma Inc., Shimadzu Corp., and Takeda Pharmaceutical
Co. These will engage in studies to advance basic research on iPS
cell technology and clinical applications, the sharing of research
materials, efficient use of public research funds, and discussion of
relevant issues with regulators.
Web site：http://www.ips-tokku.cira.kyoto-u.ac.jp/ (Japanese)
The JST Basic Research Programs
Three projects geared to expanding iPS cell research is being funded
by the Japan Science and Technology Agency (JST). These projects,
which support researchers in diverse ways, including the acquisition
of intellectual property rights, are the Yamanaka iPS cell special
project, a Core Research for Evolutional Science and Technology
(CREST) project, and a Precursory Research for Embryonic Science
and Technology (PRESTO) project.
The research project of CREST, Fundamental Technologies
The Yamanaka iPS cell special project advance research on the
The research project of PRESTO, Understanding Life by iPS
evaluation of iPS cells for cell transplantation, the pathology and the
drug screening by using patient-specific iPS cells, in cooperation
with Kyoto Unviversity, Shiga University of Medical Science, Gifu
University and Jichi Medical University.
Web site：http://y-ips.jst.go.jp/ (Japanese)
Cells Technology, aims advance technologies of cellular
reprogramming, which will contribute to understanding of
mechanisms of diseases and drug discovery. Studies by Yasuhiro
Yamada and Kenji Osafune were adopted in 2008, and Akitsu
Hotta's was accepted in 2010.
http://www.ips-s.jst.go.jp/index_e.html
46
for Medicine Concerning the Generation and Regulation of
Induced Pluripotent Stem (iPS) Cells, aims to elucidate the
mechanisms of cellular reprogramming and search new methods
for generation and differentiation of iPS cells. A research by Dr.
Haruhisa Inoue was accepted by CREST in 2009.
Web site：http://www.jst.go.jp/kisoken/crest/en/index.html
The Leading Project for the Realization of Regenerative Medicine
The Leading Project for the Realization of Regenerative Medicine
was established under the auspices of the Ministry of Education,
Culture, Sports, Science and Technology (MEXT). Under this
program, Kyoto University, Keio University, the University of
Tokyo, and RIKEN develop research activities focusing primarily
on human iPS cells with the goal of developing new therapies to
treat intractable and lifestyle-related diseases.
Web site：http://www.stemcellproject.mext.go.jp/ (Japanese)
MEXT iPS Cell Research Network
The Ministry of Education, Culture, Sports, Science and
Technology (MEXT) has developed and implemented a strategy
for the acceleration of iPS cell research. As part of this strategy, the
MEXT iPS Cell Research Network, a nationwide network of
researchers working on iPS cell studies, was established in April
2008, as a system to bring academic and research institutions
together to accelerate iPS cell research in a comprehensive manner.
Currently, more than 700 researchers are the members of the
network and share the latest information on iPS cell-related
research and intellectual property. Shinya Yamanaka is acting as
the chairman of the steering committee, and CiRA manages the
network as the secretariat.
Web site：http://www.ips-network.mext.go.jp/en/
Chairman
(FIRST Program
Cabinet Office)
Head Office
Foundation for Biomedical
Reserch and Innovation
47
Intellectual Property
Of the patent applications relating to iPS cells submitted by
Kyoto University, one was issued in September 2008, and an
additional two in December 2009. These patents cover the
following areas:
Management of iPS Cell Related
Intellectual Property at Kyoto University
Kyoto
University
1. Method for generation of induced pluripotent stem cells,
including the process of introducing of 4 transgenes -Oct3/4,
Klf4, c-Myc and Sox2 - into somatic cells.
2. Method for generation of induced pluripotent stem cells,
including the process of introducing of 3 transgenes - Oct3/4,
Klf4 and Sox2 - into somatic cells cultured with bFGF.
3. Method for generation of somatic cells by induced
differentiation of iPS cells, including processes for generating
iPS cells by introduction of 4 transgenes - Oct3/4, Klf4, c-Myc
and Sox2 - into somatic cells, or introduction of 3 transgenes Oct3/4, Klf4 and Sox2 - into somatic cells cultured with bFGF.
Under the Japanese Patent Act, these patents rights cover the
cells generated by using the methods described. The term of the
above patents is 20 years from the date of filing of the
applications, which was December 6, 2006 for all three patents.
Patents arising from CiRA research are managed by iPS
Academia Japan, Inc., which is also authorized to license their
use.
Naoko Takasu, head of CiRA Intellectual Property Management Office, and Yukinari Takao,
a deputy head of the office, check laboratory notebooks.
48
CiRA
Intellectual
Property
Management
Office
Cooperation
Office of
Society-Academia
Collaboration for
Innovation
Application of patents and
obtaining them
iPS Academia Japan, Inc.
Patent licensing
The certificate of iPS cell technology related patent,
the first one issued by the Japan Patent Office.
IP Highlights
Kyoto University obtains iPS cell patents from US biotech company,
licenses through iPS Academia Japan
On January 27, Kyoto University
acquired a number of patents (and
patents applications) on methods
for the generation of induced
pluripotent stem (iPS) cells from the
US-based biotech company iPierian
Inc., and entered into a nonexclusive licensing agreement with
iPierian through iPS Academia
Japan Inc. for a number of the
university s own iPS cell patents.
In December 2010, in order to avoid
potential future disputes over intellectual
properties relating to iPS cells, iPierian
offered to transfer its relevant patent
holdings to Kyoto University. At that
time, both iPierian and Kyoto University
had filed similar patent applications on
methods for generating iPS cells using
three defined factors to the US Patent and
Trademark Office, raising the possibility
of a dispute over priority in the following
months. The costly and time consuming
dispute would have resulted in extensive
delays as laboratory notebooks, other
documents and testimonies would have
been examined to determine rightful
ownership.
As a leading institute in the study of
iPS cells, CiRA seeks to obtain intellectual
property on related technologies so as to
ensure broad, fair, and secure access to
these cells by academic and industry
research organizations. Given the
potentially significant implications of the
issue not only for the university, but for
the entire field, Kyoto University
accepted the offer, which does not involve
payment from Kyoto University to
iPierian.
This agreement will help to prevent
the possibility of future disputes between
these two organizations over iPS cell
patents, and to create an environment
conducive to the widespread use and
study of iPS cells.
The patents transferred to Kyoto
University were based on inventions
made at Bayer Yakuhin Ltd., Kobe
Research Center which were licensed to
iZumi Bio Inc., one of two companies
which merged to form iPierian by Bayer
Schering Pharma AG in 2008. These
include a patent granted in the UK on a
method for generating iPS cells using
three defined factors. This development
specifically affects current applications in
countries around the world based on the
Japanese patent application No. 2007159382.
Kyoto University holds a press conference on February 1 to announce the patent assignment and licensing
agreements with iPierian Inc.
49
Pickup from Press Release
Safer iPS cells with L-Myc
The establishment of induced pluripotent stem cells (iPSCs)
stands as a landmark achievement in the study of cell fate
reprogramming, and has engendered tremendous hope for
applications in drug discovery and the clinic. The original iPSC
recipe, however, made use of a retrovirally-delivered factor,
c-Myc, which was shown to be associated with tumorigenicity in
mouse, raising important questions about the safety of these
cells for medical uses. A number of methods for generating
iPSCs without the use of c-Myc have subsequently been
reported, but they have been plagued by low efficiencies and
poor cell quality, and truly safe techniques for iPSC derivation
remain elusive.
All that may change with a new study by Masato Nakagawa
at the Kyoto University Center for iPS cell Research and
Application (CiRA; Shinya Yamanaka, Director), which shows
that the related molecule L-Myc can take c-Myc's place and
generate safer iPSCs at higher efficiencies. This work, published
in the Proceedings of the National Academy of Sciences, represents
an important step closer to the goal of developing clinical
applications for iPSCs in regenerative medicine.
Nakagawa began by testing the ability of all three members of
Cumulative mortality (total) (%)
100
c-Myc (47)
control (61)
L-Myc (100)
wo Myc (39)
80
the Myc family of molecules - c-Myc, N-Myc, and L-Myc - in
iPSC generation, along with the other three conventional factors,
Sox2, Oct3/4, and KLf4. In comparison to its other family
members, the L-Myc protocol showed higher efficiency in
establishing iPSC colonies, and lower tendency to generate
colonies of non-pluripotent cells. Human iPSCs generated using
L-Myc showed all the hallmarks of pluripotency, including
morphology, gene expression, karyotype, and differentiative
potency.
Generation of mouse iPSCs using L-Myc resulted in higher
ratios of colonies expressing pluripotency markers, and yielded
iPSCs with similar behavior, morphology and developmental
potential to embryonic stem cells, including the ability to
contribute to chimeras. And in perhaps the most important
difference from the c-Myc method, iPSCs generated using L-Myc
showed no increase in either tumorigenicity or mortality in
chimeric progeny. The L-Myc protocol could even be modified
to reduce the number of other factors required; Nakagawa found
that by using L-Myc, he could eliminate the requirement for
Sox2. Further studies using a number of mutant forms of c-Myc
revealed that as long the mutant's ability to promote iPSC
generation is not necessarily linked to its transformation activity,
an important discovery in the quest for safer derivation
methods.
Promotion of direct reprogramming by transformationdeficient Myc.
Proc Natl Acad Sci U.S.A. 107(32),14152-14157, 2010.
60
40
20
0
0
Cumulative mortality with tumor (%)
100
100
200
300
400
500
Observation period (days)
600
700
200
300
400
500
Observation period (days)
600
700
c-Myc (47)
control (61)
L-Myc (100)
wo Myc (39)
80
60
40
20
0
0
100
The cumulative overall mortality (Upper) and mortality with microscopically
obvious tumors (Lower) in the chimeric mice derived from iPSCs with c-Myc or
L-Myc.
50
Masato Nakagawa, Dept. of Reprogramming Science
Honors and Awards （April - December 2010）
Month
Honors and Awards
Winner
Laboratory
April
The Society of Cardiovascular Endocrinology and
Metabolism Young Investigator Award
Kohei Yamamizu
Yamashita Lab
TOKYO Techno Forum 21 The 16th Gold Medal Award
Kazutoshi Takahashi
Takahashi Lab
The Mayor of Osaka Special Award
Shinya Yamanaka
Yamanaka Lab
The 2010 March of Dimes Prize in Developmental Biology
Shinya Yamanaka
Yamanaka Lab
The Japanese Orthopaedic Association The extraordinary
members acting as academic consultant for JOA
Shinya Yamanaka
Yamanaka Lab
The 100th Imperial Prize and Japan Academy Prize,
The Japan Academy
Shinya Yamanaka
Yamanaka Lab
The 8th International Society for
Stem cell Research (ISSCR) Travel Award
Kumiko Iwabuchi
Okita Lab
The 8th International Society for
Stem cell Research (ISSCR) Travel Award
Kohei Yamamizu
Yamashita Lab
The 16th International Vascular Biology Meeting (IVBM) Kohei Yamamizu
Travel Award
Yamashita Lab
Travel Award, the Global COE Project "Center for Frontier
Medicine", Kyoto University Graduate School of Medicine
Hideki Uosaki
Yamashita Lab
July
The 8th Metabolic Syndrome Conference Young
Investigator Award
Kohei Yamamizu
Yamashita Lab
August
Best Presentation Award, the 31st annual meeting of the
Japanese Society for Inflammation and Regeneration
Hideki Uosaki
Yamashita Lab
October
The Kyoto Medal of Honor
Shinya Yamanaka
Yamanaka Lab
Tohoku University the Best Graduate Student Research Award
Takafumi Toyohara
Osafune Lab
The 2010 Medical Award of the Japan Medical Association
Shinya Yamanaka
Yamanaka Lab
The 2010 Person of Cultural Merit
Shinya Yamanaka
Yamanaka Lab
The 26th annual Kyoto Prize in Advanced Technology
Shinya Yamanaka
Yamanaka Lab
The 2010 Balzan Prize for Stem Cells: Biology and
Potential Applications
Shinya Yamanaka
Yamanaka Lab
AHA BCVS International Travel Grant
Hideki Uosaki
Yamashita Lab
The 18th Japanese Vascular and Medicine Organization YIA
Kohei Yamamizu
Yamashita Lab
May
June
November
December
51
CiRA in the Media （April - December 2010）
Month
April
May
Subject
Kazutoshi Takahashi receives the Gold Medal Award
Shinya Yamanaka introduced on the
Japan Patent Office website
Shinya Yamanaka talks about CiRA's mission in the
column, "Power of Kansai"
Profile of Kazutoshi Takahashi, the winner of the
Gold Medal Award
Jun Yamashita's team reports new insights into the
mechanisms of arterial development
CiRA publishes its newsletter, Vol. 1
Shinya Yamanaka stranded in Sweden due to the
volcanic eruption in Iceland
Feature "iPS cells, now" part 1
Researchers from CiRA and other institutes report
issues arising from Japanese regulations
iPS Academia Japan enters into licensing
agreements with CDI, a US biotech venture
Ceremony held to celebrate the launching of CiRA as
an independent institute
Shinya Yamanaka comments on draft revised
guidelines for clinical research using iPS cells
CiRA starts a research project to develop
new drugs for rare diseases,
Takeda Pharmaceutical offers cooperation.
Stem cell therapy explained in a column,
"Curious word"
Features of the new CiRA facilities
June
July
Shinya Yamanaka and other two professors
receive the 2010 Kyoto Prize
iCeMS and CiRA to hold a workshop for high school
students in August
Hideyuki Okano of Keio University and Shinya
Yamanaka report that safer iPS cells were
effective in restoring function in mouse model of
spinal cord injury
Shinya Yamanaka seeks to build
the best iPS cell research institute in the world
No. 1 scientists in Japan
Masato Nakagawa reports the generation of
safer iPS cells with L-Myc
All-Japan system to advance iPS cell
research starts moving
CiRA launches as an independent institute
52
Media
Yomiuri Shimbun
Nikkan Kogyo Shimbun
Yomiuri Shimbun
Yomiuri Shimbun
Nikkei Shimbun, Sankei Shimbun
Yomiuri Shimbun
Yomiuri Shimbun
Yomiuri Shimbun
Kyoto Shimbun, Mainichi Shimbun,
Kyodo News,Nikkan Kogyo Shimbun,
Sankei Shimbun, Yomiuri Shimbun
Asahi Shimbun
Mainichi Shimbun, Yomiuri Shimbun,
Sankei Shimbun, Nikkei Shimbun, Kyoto Shimbun
Nikkei Shimbun
Kyoto Shimbun, Mainichi Shimbun,
Yomiuri Shimbun,
Sankei Shimbun, Chunichi Shimbun
Yomiuri Shimbun
Nikkei Shimbun
Yomiuri Shimbun
Kyoto Shimbun
Yomiuri Shimbun
Kyoto Shimbun
Mainichi Shimbun
Yomiuri Shimbun
Yomiuri Shimbun
Yomiuri Shimbun
Nikkei Shimbun, Yomiuri Shimbun, Asahi Shimbun,
Mainichi Shimbun, Kyoto Shimbun, Chunichi Shimbun
Yakuji Nippo
Yomiuri Shimbun, Mainichi Shimbun
Kyoto Shimbun, Sankei Shimbun
Nikkei Sangyo Shimbun, Sankei Shimbun,
Mainichi Shimbun, Nikkan Kogyo Shimbun
Kyoto Shimbun, Nikkei Shimbun,
Chemical Daily
Osaka Nichinichi Shimbun
Yakuji Nippo
Yomiuri Shimbun
NHK
Release
4.15
4.16
4.20
4.18
4.21
4.23
4.23
4.26
5.3
5.7
5.14
5.8
5.10
5.8
5.9
5.10
5.9
5.9
5.10
5.10
5.15
5.18
5.25
5.17
5.24
5.31
6.19
6.21
6.25
7.4
7.6
7.7
7.8
7.9
7.16
7.26
7.15
Weekly Gendai (Kodansha) 7.17
Sankei Shimbun, Mainichi Shimbun, Yomiuri
Shimbun, Asahi Shimbun, Nikkei Shimbun,
7.27
Nikkan Kogyo Shimbun, Nikkei Sangyo Shimbun,
Chemical Daily, Kyoto Shimbun, Osaka Nichinichi Shimbun
Nikkei Science(Nikkei Science)
July,2010
Health & Beauty (Kodansha)
July,2010
Month
August
Subject
iCeMS and CiRA hold a workshop for
high school teachers
Reportage: A workshop for high school teeachers
organized by iCeMS and CiRA
iPS Academia Japan concludes a licensing
contract with Axiogenesis AG of Germany
CiRA to hold a symposium for the
gereral public in Tokyo on Oct. 2
September Anchorman Junichi Sumi visits the most advanced
institute at Kyoto University (Chichin Puipui)
Shinya Yamanaka to receive the Balzan Prize
Hiroshi Egusa of Osaka University and
Shinya Yamanaka's grpup generate iPS cells from
human gum tissue
The man who changed the future of ``life"
∼ Shinya Yamanaka・iPS cell revolution ∼
Shinya Yamanaka to receive the Kyoto Medal of Honor
October
iPS Academia Japan launches business to
supply iPS cells to private organizations
Takashi Tachibana closes in on iPS cells
An interview with Shinya Yamanaka
CiRA holds a symposium in Tokyo
The city of Kyoto presents Shinya Yamanaka
the Kyoto Medal of Honor
Shinya Yamanaka to receive the Kyoto Prize
iPS Academia Japan grants French biotech firm
Cellectis the rights to iPS cell technology-related
patents
What is true courage? Kobe Steel Rugby Club
GM Seiji Hirao vs Shinya Yamanaka
CiRA announces a collaborative project with
French biotech firm Cellectis
"All for patients" Shinya Yamanaka
Shinya Yamanaka selected as one of the Persons of
Cultural Merit
November
December
The face of Japan: Shinya Yamanaka
Elementary school pupils interview Takuya Yamamoto
Child reporters from a NHK program
interview Megumu Saito
Shinya Yamanaka awarded the Kyoto Prize
Shinya Yamanaka starts drawing safety evaluation
standards toward building an iPS cell bank
Shinya Yamanaka gives a lecture at a workshop
commemorating the Kyoto Prize
Shinya Yamanaka gives a special class during the
Kyoto Prize Forum for high school students
The Balzan Prize award ceremony held at the
presidential palace in Italy
An interview with the winner of the Kyoto Prize
The Yamanaka-Balzan Award set up
Kyoto bizW: Turning Point
Nuclear reprogramming selected as one of major 10
research results in the past decade: Science
CiRA to set up an iPS cell bank
Science Zero Special: Scientific News 2010
Media
KBS Kyoto
Kyoto Shimbun
Mainichi Shimbun
Chunichi Shimbun
Release
8.2
8.3
8.25
8.5
Nikkei Sangyo Shimbun, Sankei Shimbun, Mainichi
8.4
Shimbun, Nikkan Kogyo Shimbun, Nikkei Shimbun,
Chemical Daily
8.5
Mainichi Shimbun
8.10
MBS
9.1
Kyoto Shimbun, Yomiuri Shimbun, Asahi Shimbun
Nikkei Shimbun, Mainichi Shimbun, Japan Times
Mainichi Shimbun, Nikkei Shimbun, Kyoto Shimbun,
Yomiuri Shimbun, Asahi Shimbun
Nikkan Kogyo Shimbun, Sankei Shimbun, Nikkei
Sangyo Shimbun
NHK
9.7
9.8
Yomiuri Shimbun, Mainichi Shimbun
Asahi Shimbun, Sankei Shimbun, Nikkan Kogyo
Shimbun
Yomiuri Shimbun, Nikkan Kogyo Shimbun, Nikkei
Sangyo Shimbun
Bungeishunju
Chunichi Shimbun
Nikkei Shimbun, NHK
Nikkei Shimbun, Kyoto Shimbun
Asahi Shimbun, Mainichi Shimbun, Sankei Shimbun
Japan Times
Nikkei Shimbun, Mainichi Shimbun, Sankei
Shimbun, Yomiuri Shimbun
Asahi Shimbun
Weekly Gendai (Kodansha) 9.29
Nikkei Shimbun, Sankei Shimbun
Nikkei Business (Nikkei Business Publications)
Mainichi Shimbun, Yomiuri Shimbun, Kyoto
Shimbun, Nikkei Shimbun, Sankei Shimbun
Asahi Shimbun, Nikkan Kogyo Shimbun
Bungeishunju
Chunichi Shimbun
NHK
Yomiuri Shimbun, Sankei Shimbun, Kyoto Shimbun
Nikkan Kogyo Shimbun, Nikkei Shimbun
Nikkei Shimbun
Kyoto Shimbun
Kyoto Shimbun, Nikkan Kogyo Shimbun
Mainichi ｊｐ , La Stampa, Il Sole 24 Ore,
Corriere della Sera
Asahi Shimbun
Yomiuri Shimbun, Kyoto Shimbun, Asahi Shimbun,
Nikkei Shimbun, Nikkan Kogyo Shimbun
KBS Kyoto
Yomiuri Shimbun, Nikkei Shimbun, Jiji Press,
Kyodo News
Kyoto Shimbun, Yomiuri Shimbun, Nikkei Shimbun
NHK
9.15
9.16
9.18
9.30
9.30
September,2010
10.1
10.3
10.15
10.16
10.18
10.18
10.19
10.23
10.25
10.26
10.26
10.27
October,2010
11.7
11.7
11.11
11.16
11.16
11.17
11.19
11.23
12.2
12.3
12.19
12.23
12.25
53
Events
Activities for researchers
Lectures and practical training courses
CiRA hosts lectures and training programs on the
generation, maintenance, and culture of iPS cells. Two
lectures and five training courses were held during fiscal 2010.
Lectures on Human iPS Cell Generation and Maintenance
Date
2010.8.30
2011.1.31
Title
1st lecture on the generation and
maintenance of human iPS cells
2nd lecture on the generation and
Training courses on the Generation and Maintenance of Human iPS Cells
Date
2010.6.28-30
Title
1st training course on the generation and
2010.8.30-9.1
2nd training course on the generation
and maintenance of human iPS cells
2010.11.29-12.1 3rd training course on the generation and
54
Date
2011.1.26-28
2011.3.23-25
Title
4th training course on the generation and
5th training course on the generation and
CiRA Seminars
CiRA holds invited seminars by scientists from around the world to share their latest research results.
Seventeen such talks were held between April and December 2010.
CiRA seminar 2010
Date
Title
Speaker
4.26
Direct Reprogramming of Cardiac Fibroblasts
into Functional Cardiomyocytes by Defined Factors
Masaki Ieda
5.28
The Pharmaceutical Affairs Act and Products created from Biological Materials
Yoshinobu Hirayama
7.14
Modeling leukemogenesis and hematopoiesis in transgenic mice and zebrafish
Pu Paul Liu
7.27
Issues to Promote Translational Research
Satoshi Toyoshima
8.26
Transcriptional and epigenetic regulation of T cell differentiation
John Joseph O Shea, Jr.
9.22
From Development to Pathology – Hedgehog Signaling
in Osteoarthritis and Chondrosarcoma
Benjamin Alman
10.12
Stem cell tourism: Treatments with little scientific-basis
Douglas Sipp
10.14
Cell therapy for Parkinson's disease - Clinical perspectives
Deniz Kirik
10.14
Cell therapy for Parkinson's disease - Reporter mice as research tools
Lachlan Thompson
10.14
Cell therapy for Parkinson's disease - WNT signaling and DA neurons
Clare Parish
10.21
Expansion and Differentiation of Pluripotent Stem Cells in Stirred Suspension
Derrick E. Rancourt
11.2
FGF-Erk signaling in pluripotency and lineage commitment
Tilo Kunath
11.4
Communication Skills to Activate Organizations – E-mail, Conference and Dialogue –
Takayuki Shiose
11.29
Transient activation of c-MYC expression is critical for efficient platelet generation
from human induced pluripotent stem cells: heterogeneity of iPS cells reveals molecular
implication of normal platelet generation
Koji Eto
11.30
Directed induction of chondrogenic cells from mouse adult dermal fibroblast culture
by defined factors
Noriyuki Tsumaki
12.1
Engineering pluripotent stem cell differentiation to lineage-specific chondrocytes
Naoki Nakayama
12.20
Role of CD44v in cancer stem cells and metastasis
Hideyuki Saya
55
Events
Activities for the general public
The grand opening of the CiRA research building and inaugural ceremony（May 8）
CiRA held an opening ceremony on May 8 to celebrate its establishment as
an independent institute on April 1, 2010, and the grand opening of the new
research building. Nearly 350 people, including government officials,
lawmakers, researchers, and representatives from patients groups
participated in the event.
iCeMS/CiRA Classroom 2010：Hands-on with Stem Cells!
（Aug. 4-5）
CiRA and iCeMS, the Institute for
Integrated Cell-Material Sciences,
organized a two-day workshop for
32 high school students on August
4 and 5. The educational program
gave participants the opportunity
to experience working with ES and
iPS cells.
56
CiRA Symposium 2010 The Frontline of iPS Cell Research
（Oct. 2）
CiRA hosted a symposium for the general public in Tokyo on October 2. Some
650 people, including patients with intractable diseases and their families,
took part in the meeting, in which Shinya Yamanaka, Jun Takahashi, and
Kenji Osafune gave lectures on their latest research activities.
CiRA Ground Floor Activity（Oct. 18-29）
The CiRA Ground Floor Activity Part
1 took place at the gallery space
within the CiRA building between
October 18 and 29, featuring
science illustrations by Tomoyuki
Narashima, an illustrator working in
the United States.
57
Publications
① CiRA Brochure (Japanese)
The CiRA Brochure provides an
overview of the institute s
organization, history, intellectual
property, and research.
② CiRA Newsletter (Japanese)
The quarterly newsletter reports
research and other activities at
CiRA, and publishes interviews
and responses to frequently
asked questions to help the
general public gain a clearer
understanding of iPS cell
research.
③ Stem Cell Handbook – Cells that Replicate Themselves in the Human Body
This 12-page booklet provides
basic information about stem
cells, such as stem cell properties,
the differences between ES and
iPS cells, and social issues in stem
cell research.
These publications can be downloaded via the CiRA website at no cost,
and are distributed to visitors to the CiRA gallery and participants at events sponsored by CiRA.
58
(Japanese)
Operation
Fiscal 2010 Budget
iPS Cell Research Fund
(Donations)
CiRA s budget for fiscal 2010 ending in
March 31, 2011, stood at 4.18 billion yen
– 3.8 billion yen in research grants from
government agencies, 270 million yen in
basic operating funds, also from the
government, 70 million yen from the
private sector, and 40 million yen in
donations.
(As of Dec. 31,2010)
40
Private sector grants
70
Basic operating funds 270
Grants-in-Aid
for Scientific
Research
660
Total
Other public
research grants
710
4,180
（million yen）
FIRST grant
(Cabinet Office)
2,430
CiRA Staff
Approximately 150 people, including
faculty members, technicians and
research support and administrative staff,
were working at CiRA, as of March 1,
2011. In addition, dozens of graduate
students are carrying out research
activities in 19 laboratories.
Professor …………………………………………………………… 6
Associate Professor …………………………………… 5
Lecturer ……………………………………………………………… 6
Assistant Professor …………………………………… 6
Postdoctoral Fellow
………………………………
Research Support Staff
………………………
Research Strategy Division
Administration Division
Total
(As of March 1, 2011)
39
58
………………
21
………………………
12
153
59
Since the establishment on January 22,
2008 of the Center for iPS Cell Research
and Application within the Kyoto
University Institute for Integrated CellMaterial Sciences (iCeMS), which later
became the independent institute of the
same name (CiRA), we have enjoyed
strong support from a great many people.
On April 1, 2009, we established the
iPS Cell Research Fund to further
strengthen our research activities. We
would like to show our deep appreciation
to all those who made contributions to
CiRA. The names of the donors who gave
us prior consent as of December 1, 2010,
are listed.
Donors
Fiscal 2008
Fiscal 2009
Kyoko Okubo
Yukiko Shirakawa
Kayoko Abe
Takashi Ono
Tetsutaro Yasuhira
Hideyuki Aiura
Hiroko Ooishi
Daiwa Securities
Group Inc.
Yoshiko Amano
Toru Osako
Yasuko Bitou
Hisae Saito
Katsuyuki Hara
Fujiko Saitou
Sachiyo Hara
Noboru Sakaguchi
Soichiro Hara
Fukuko Sano
Kumiko Hirata
Tetsuhiko Sasaki
Satiko Horibe
Kazuo Shiimoto
Tetsuya Hoshikawa
Yasuko Suga
Sadako Ichihara
Kazuko Takahata
Sonoko Inoue
Yumiko Takeda
Aiko Ishihara
Yoshihiko Takeda
Yuki Ishihara
Kikuno Tanaka
Hisako Iwahashi
Kenji Tanaka
Makoto Kaji
Kenji Toyoda
Toyoko Kato
Yoshiyuki Uchida
Katuyoshi Kawano
Yasuko Ueda
Toshiko Kodaka
Toshihiko Urakubo
Kazumi Kosui
Itue Watanabe
Toshiko Maeji
Hiroko Yokoyama
Yoshie Matsuoka
Shigeru Yoshida
Mihoko Miyake
Atsuo Yoshimura
Masakazu Mizutori
Takako Yoshizawa
Haruko Morita
Akiko Yurugi
Hisako Moriyama
Nihon Karuta MFG, Co.LTD
Satoshi Mukaigawa
Saitama Parkinson's
Disease Association
Sumitomo Mitsui Banking
Corporation
Anonymous /
Unanswered 5
Yukiko Nakane
Yoichi Niozu
Sumitomo Mitsui
Banking Corporation
Kyoko Nishida
Taiheido Corporation
Kazushi Nomura
USACO Corporation
Akie Ogawa
Anonymous /
Unanswered 33
Akiyo Ōiwa
A list of donors names is displayed near the entrance on the first floor of the CiRA
research building.
60
Shigeharu Okoshi
Fiscal 2010
Hiroko Wakasa
Takayuki Awasaki
Ikumi Yamamoto
Masanori Imaizumi
Apoburēn Center
Yoshinobu Ito
Bank of Kyoto, Ltd.
Shigeru Izawa
Nagata Pharmaceutical
Co., Ltd.
Morito Kamata
Shuhei Katori
Yasuhiro Kawahara
Mototsugu Kojima
Ken Kougo
Yasuo Kumazawa
OHKI Corporation
Retina Degeneration Research
Fund (MOUMAKU-KIKIN)
TAKASHIMA
International Patent Office
Anonymous /
Unanswered 50
Shiro Kuniya
Setsuko Minamitani
Fumio Mizoguchi
Takahide Mori
Shinya Moriwaki
About the
■ Purpose
iPS cell technology has great potential for uses in
the development of applications for drug
discovery and therapeutics, but a number of issues
must first be solved. To make the promise of iPS
cells possible, we are striving to secure talented
human resources from around the world, acquire
intellectual property and establish a stable
financial base. All donations to the iPS Cell
Research Fund are used to achieve the purposes.
■ Merits of donation
Upon obtaining prior consent, donor names are
displayed with a message of gratitude on the wall
of the CiRA entrance hall. Donors also receive the
CiRA newsletter, brochure, our Stem Cell
Handbook, and other publications, as well as
notifications regarding upcoming CiRA events.
■ Tax deductions
Donation to the research fund can be claimed as
exemptions under the Japanese personal and
corporate income tax laws.
Hisao Mouri
Tadaaki Nakagawa
Hirohisa Nakata
Donations from individuals
Donations of 2,000 yen and over, up to a total of
40% of income for the tax year, can be deducted.
Please submit a Donation Receipt as a separate
attachment when sending the donation form. For
residents of Kyoto City and Kyoto Prefecture, the
prefectural tax of 4% and city residential tax of
6% on donations of 5000 yen and over, up to 30%
of income for the tax year, can also be deducted.
Yasuzo Niwa
Kazushi Nomura
Kaori Okano
Takeo Onishi
Akiko Ono
Hiroaki Sakae
Donations from businesses
The full amount donated can be claimed as
deductible expenses.
Hiroshige Sayo
Yutaka Shirai
Shigeyuki Sugito
Sachiko Takada
Yoshihiko Takeda
Kunihiro Tatsukawa
Hiroyuki Tsuji
Yoshiyuki Uchida
Yasuko Ueda
Note: Upon obtaining prior
consent, names of donors to the
iPS Cell Research Fund and others
who made donations to CiRA in
other capacities are listed. Donors
who have not responded to
requests for permission to publish
their names are included in the
Unanswered category.
Contact
Secretariat, iPS Cell Research Fund
TEL: +81 75 366 7000 FAX: +81 75 366 7023
CiRA the iPS Cell Research Fund
(Japanese language only)
http://www.cira.kyoto-u.ac.jp/e/about/fund.html
Tsuyoshi Ueki 61
Glossary
C
β-catenin signaling
acquired chemical modifications (DNA
methylation and histone modification) to
chromatin, a complex of DNA and proteins.
Epigenome
This word refers to all the epigenetic
modifications that have occurred within a
certain cell (DNA methylation, histone
modification, etc.).
β-catenin is an important component of the
well-known Wnt signaling pathway. The Wnt/
β-catenin pathway is widely involved in the
regulation of cell fate determination, cell
proliferation, and control of development in
vertebrates and invertebrates.
Chimeric mouse
Chimeras are formed from two or more
embryos with different genomes or a part of
them. For example, a mouse created by
transplanting iPS cells or ES cells into an early
embryo is called a chimeric mouse.
ES cells (embryonic stem cells)
ES cells are a type of pluripotent stem cells
derived from the inner cell mass of the
blastocyst six or seven days after fertilization
and culturing them; they can differentiate into
cells of any tissue in the body. However, it has
been noted that immune rejection will be a
problem in cell transplantation therapy as the
cells cannot be created from those of the
patient s own body; ES cells are obtained by
destroying the embryos.
F
Clone
In the biological sense, populations that have
the same genetic information are called clones.
Population of cells with the same genetic
information grown from a single cell is called
clones or cell lines.
Cyclic AMP
A compound that acts as an intracellular
mediator for intracellular signaling, is involved
in gene regulation, and has an important role in
various biological functions.
Feeder cells
Cells with a complementary role in adjusting
culture conditions when culturing target cells.
They are usually chemically treated to prevent
division. In iPS cell cultures, fibroblasts derived
from mouse fetuses are used as feeder cells.
E
Epigenetic mechanism
A mechanism where changes in phenotype and
the amount of gene expression are caused by
mechanisms other than changes in the
underlying DNA sequence. It occurs by
62
High-throughput screening
A technology whereby target compounds are
selected from catalogs containing a wide variety
of compounds (compound libraries) using
automated devices such as robots.
Homologous recombination
technology
Homologous recombination is recombination
occurring in regions of similar DNA sequence
(homologous sites). A technology that exploits
the ability of double-stranded DNA to repair
itself based on the complementary strand even
when cleavage or mutation occurs to alter
genetic information at target locations.
I
Flow cytometry
Light scattering and fluorescence measurements
using lasers allow the analysis of biological
characteristics of cells such as the size of single
cells passing through water and their DNA
content.
Full-length cDNA
A complementary strand of DNA copied
completely from the base sequence and the
information in mRNA is transcribed during
protein synthesis only from regions functioning
as a gene (coding). Since it contains the design
information required to synthesize a full-length
protein, full-length cDNA can synthesize a fulllength protein.
iPS cells (induced pluripotent stem cells)
iPS are a type of pluripotent stem cells
established by introducing specific factors into
somatic cells and are similar to ES cells. The
world s first successful establishment through
the work of Prof. Yamanaka using somatic cells
from mice was reported in 2006.
K
G
Embryoid body (EB)
When iPS and ES cells are cultured in
suspension, they form ball-shaped cell clusters.
When cultured in this state for approximately 2
weeks, differentiation into various cell types is
observed. This is a commonly used means of
examining the differentiation of pluripotent
cells.
H
In vitro and in vivo
In vitro is a term that refers to experiments
performed under environmental conditions
similar to those within a test tube under
predetermined experimental conditions, and in
vivo is a term that refers to experiments
performed within the body of a laboratory
animal such as a mouse.
D
DNA methylation
In mammals, this refers to the replacement of
hydrogen (-H) by a methyl (-CH3) group at the
5 position of cytosine. Methylation of the
region regulating the working of the gene is
known to inhibit gene expression, whereas
removal of the methyl group (demethylation) is
known to activate gene expression. A
mechanism for gene expression independent of
changes in base sequences (epigenetics).
Germline transmission
Differentiation of pluripotent stem cells into
germline cells and the transmission of the
genetic information derived from the
pluripotent stem cells to the next generation
through chimeric mice or the like. Chimeric
mice in which iPS cells contribute to the
development of the whole body are born from
descendants of individuals in which germline
transmission occurred.
Genome
The entire genetic code of an organism is called
its genome. Typically, the genome is described
on the basis of the organism s species and called
the human genome in the case of humans and
mouse genome in the case of mice.
Germ layers
The mass of cells formed from embryos after
fertilization and divided into the endoderm,
mesoderm, and ectoderm. The endoderm later
forms the respiratory and digestive organs. The
mesoderm differentiates into bone, cardiac
muscle, and erythrocytes. The ectoderm forms
the nervous and sensory organs.
Karyotype
The chromosomal constitution represented by
the number, size, and form of chromosomes in
the organism.
L
Low-molecular-weight compound
Among certain low-molecular-weight
compounds with physiological functions such
as enzyme inhibitory activity, valproate, which
are effective in promoting the establishment of
iPS cells, have been found.
M
Microarray
A technique allowing the exhaustive testing of a
huge quantity of DNA or protein at the same
time.
N
Neurosphere
A spherical mass of neural cells generated from
neural stem cells. The primary neurosphere is
formed by differentiation from ES and iPS cells,
and its further subculture is called the
secondary neurosphere. It is used for
subculturing neural stem cells in suspension
culture.
Next-generation sequencer
Using a next-generation sequencer, in addition
to decoding large amounts of genomic DNA
sequences at high speed, it is possible to detect
RNA with a variety of functions, identify and
detect the distribution of transcription start
sites in the genome, and detect DNA–protein
interactions at high speed.
Notch signaling
A cell signaling system for regulation of cell fate
decision during development observed in stem
cells, and also present in most multicellular
organisms. It is involved in diverse regulation
regarding the determination of cell fates,
particularly in the development of nerves, heart,
and endocrine glands.
P
p53
The p53 gene is a typical tumor suppressor
gene. It has important roles such as maintaining
cell homeostasis and inducing apoptosis (cell
death). For a cell to become cancerous, changes
are needed in multiple oncogenes and tumor
suppressor genes, but p53 gene abnormalities
are the most frequent in malignant tumors.
Positron emission
tomography–CT (PET–CT)
A device that uses a special camera to take
tomograms of the biodistribution of a drug
labeled with an isotope that emits positivelycharged electrons and injected into the body. It
is a useful diagnostic equipment for examining
the type of cancer.
Q
QT time
QT time is the average length of the action
potential duration (APD) in a ventricular
muscle. It shows the state of activity of
ventricular muscle cells.
R
Random integration
This terminology refers to the introduction of
foreign genes at unspecified locations on the
chromosome.
Reprogramming
The resetting of the nucleus of a differentiated
somatic cell, its reversion to the state of a cell
nucleus in the early stages of development such
as the nucleus of a fertilized egg, and its
transformation into a pluripotent stem cell.
Retroviral vector
Vectors act as carriers in the introduction of
external genes into the cell. Vectors derived
from viruses have been actively developed
because of their high gene transfer efficiency.
The target gene is incorporated into a virus and
the gene is introduced by infecting the cell.
Retroviral vectors are a type of virus vectors
with the ability to infiltrate and proliferate their
own DNA within the host cell s DNA after
infecting the host cell, and because of this
ability, they are used as vehicles for introducing
genes.
Stem cells
The human body is made up of
approximately 60 trillion cells, some of which
can be replenished and are called as stem
cells. Stem cells can divide and
simultaneously produce a cell similar to the
original and a cell that will differentiate into
another cell. Stem cells are of various types
such as those present in the body such as
neural stem cells, epithelial stem cells, liver
stem cells, germline stem cells, and
hematopoietic stem cells and those that are
artificially produced such as iPS and ES cells.
Striatum
A subcortical structure of the telencephalon,
one of the major components of the basal
ganglia.
Subculture
It is made by transferring cells from a
previous culture to a fresh growth medium.
T
Teratoma
When ES and iPS cells are subcutaneously
injected into immunodeficient mice, tumors
form. These tumors, called teratomas, are
mixtures of various types of tissues.
Observing teratomas and confirming that
they have differentiated into various tissues is
a common way to examine the differentiation
of pluripotent cells.
Transcriptome
This term refers to the complete set of
primary transcripts such as mRNA present in
a single cell. While the genomes of cells in the
same individual are essentially identical,
transcriptomes differ in cells and tissues with
different functions since genes function
differently depending on the tissue even in
the same individual.
S
piggyBac transposon
A transposable genetic element. Chromosome
translocated from one part to another.
When genes are introduced into the cell using
this transposon, genomic insertion of foreign
genes can be avoided.
Plasmid vector
A plasmid vector is introduced into the host cell
by methods such as reagents or electroporation
and causes the extrachromosomal expression of
foreign genes. Transfer efficiency is generally
believed to be higher for virus vectors such as
retroviral vectors. Plasmid vectors are stable and
can be stored for a long time after preparation.
They can also be created in an ordinary
laboratory.
SNP array
Locations where one base has been replaced by
another although the underlying genetic
sequence is identical are called SNPs (single
nucleotide polymorphisms). Differences in
SNPs are believed to produce individuality in
physical characteristics. A SNP array is an
experimental tool used to detect SNPs.
Somatic cell
A generic non-germline body cell.
Splicing
The mRNA precursor transcribed from typical
eukaryotic DNA contains regions called introns,
which are not directly connected to protein
amino acid sequence. The process of excluding
these introns and producing mRNA comprising
the remaining regions known as exons is called
splicing.
Transgene
This term refers to DNA (or genes) stably
introduced and inherited by individuals in
the next generation.
U
Undifferentiated cell markers
Genes specifically expressed in
undifferentiated ES and iPS cells. The fact
that these genes are expressed indicates that
the cells are in an undifferentiated state.
Conversely, various differentiated cell
markers are also present in differentiated cells
such as nerves, muscles, and blood.
63
CiRA Facilities
The CiRA facilities on the Kyoto
University Yoshida campus were
completed in February 2010.
The 12,000 square meters building,
with five aboveground levels and one
basement floor, accommodates a cell
processing center and a laboratory
animal facility.
The gallery on the first floor is
open to the public on weekdays.
Address
53 Kawahara-cho, Shogoin, Sakyo-ku,
Kyoto 606-8507, Japan
Access
●
From Kansai International
Airport to Kyoto
Take JR "Haruka" Kansai Airport Limited
Express from Kansai airport,
get off at Kyoto station
JR (Japan Railway) Line
Shinkansen Line
阪急電車
From Kyoto station to CiRA
Take the bus No.206 "bound for
Higashiyama St. and Kitaoji Bus
Terminal", get off at Kumano Jinja-mae
N
Keihan Demachiyanagi Sta.
琵琶湖
Lake Biwa
梅田
Umeda
近衛通 Konoe St.
南西病棟
再生医科学研究所
（西館）
South West Wards
Institute for Frontier
Medical Sciences
(West Bldg.)
京阪神宮丸太町駅
Keihan Jingu-Marutamachi Sta.
関西国際空港
Kansai Int’l Airport
64
天王寺
Tennoji
Kyodai Seimon-mae
近衛通
Konoedori
病院構内
University
Hospital
再生医科学研究所
（東館）
Institute for Frontier
Medical Sciences(East Bldg.)
大阪
Osaka
Osaka Bay
京大正門前
Higashi -Oji St.
三宮
Sannomiya
大津
Otsu
百万遍
Hyakumanben
東大路通
京都
Kyoto
駅
Train Station
今出川通 Imadegawa St.
Marikoji St.
河原町
Kawaramachi
新大阪
Shin-Osaka
Kawabata St.
Kyoto City Subway
Kamogawa River
神宮丸太町
Jingu-Marutamachi
烏丸御池
Karasuma Oike
三条京阪
Sanjo Keihan
京都市営地下鉄
鴨
川
バス停
Bus Stop
鞠小路
Keihan Railway
京阪出町柳駅
川端通
出町柳
Demachiyanagi
Hankyu Railway
京阪電車
大阪湾
●
Yoshida Campus,
Kyoto Univ.
新幹線
新神戸
Shin-Kobe
From Tokyo to Kyoto
Take JR "Nozomi" or "Hikari"
Shinkansen bullet train bound for
"Hakata" or "Shin-Osaka" at
Tokyo station, get off at Kyoto
京都大学
吉田キャンパス
N
JR
●
iPS細胞研究所
Center for iPS Cell Research
and Application(CiRA)
丸太町通 Marutamachi St.
春日通 Kasuga St.
熊野神社
Kumanojinja
熊野神社前
Kumanojinja-mae
Publisher
Center for iPS Cell Research
and Application (CiRA), Kyoto University.
Producer
Masahiro Kawakami, Akemi Nakamura (CiRA)
Production cooperation
Saki Tamura, Sumie Minakuchi (CiRA)
Kazuyuki Kamano, Miki Fukuda, Yoshihito Fujimoto
Mari Watanabe (CiRA)
Douglas Sipp
The texts of the ``Research Groups’’ (page 8 – 45)
were written by investigators at each laboratory.
Editor
Ayumi Kojima
Photography
Jussi Panula (CiRA)
Photographs (Cover page & p3):
Tetsuhiro Kikuchi, Kazutoshi Takahashi
Shinya Yamanaka (CiRA)
Design
Hiroaki Yasojima, Daisuke Inoue (GRID CO., LTD)
Print
SHINKOSHA CO.
Publication date March 31, 2011
Center for iPS Cell Research and Application,
Kyoto University.
53 Kawahara-cho, Shogoin, Sakyo-ku,
Kyoto 606-8507, Japan
Phone +81-75-366-7000
Fax
+81-75-366-7023
Email ips-contact@cira.kyoto-u.ac.jp
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H2

CiRA Annual Report2010 - 京都大学iPS細胞研究所 CiRA（サイラ）

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