基礎研究から臨床応用へ：ES細胞、iPS細胞を用いたパーキンソン病に対する細胞

脳神経疾患における再生医療
講演１
セッションⅢ
基礎研究から臨床応用へ：E S 細胞、iP S 細胞を用いた
パーキンソン病に対する細胞移植治療の将来展望
期待できる。この治療法は、ドーパミン神経をドナ
ー細胞として、ドーパミンの不足した線条体に移植
するという方法である。1987年から約400人のパー
キンソン病患者さんに、胎児組織を用いた細胞移植
治療が行われて来た。いくつかの open label の臨床
試験では劇的な効果が得られる症例も報告された。
最近では移植手術を受けた患者さんの剖検例から、
術後10年以上経過しても移植したドーパミン神経が
京都大学再生医科学研究所生体修復応用分野研究員
森実飛鳥
略　歴
1996　東京医科歯科大学医学部医学科卒業
2004　京都大学大学院医学研究科博士課程卒業
博士学位（京都大学医学博士）授与
2004　神戸市立中央市民病院脳神経外科勤務
2004　日本脳神経外科専門医修得
2005　日本脳神経血管内治療学会専門医修得
2006　スウェーデン　ルンド大学（研究員）留学
パーキンソン病に対する細胞移植治療の研究に従事
2008　京都大学再生医科学研究所生体修復応用分野研究員
脳の中で生き続けていた事が報告された。一方、ル
ビー小体というパーキンソン病の病理学的な変化
が、これらの移植片の中でも見られたと報告されて
いる。さらにNIH主導で行われた２重盲検試験では、
残念な事にプライマリーエンドポイントで移植の有
効性が示されなかった。胎児中脳移植を用いた移植
の主な問題点は、均質な標準化されたドナー細胞を
得る事が、供給量の面からも難しいという事である。
さらにこの方法では中絶胎児の組織を用いるため、
幹細胞（stem cell）が再生医療の有力な道具に
倫理的問題が常につきまとう。
成りうるとして注目を集めている。ターゲット疾患
以上のような背景に加え、近年の幹細胞研究の目
の一つとしてパーキンソン病がとり挙げられる事が
覚ましい進歩により、次世代の細胞ドナー資源とし
多い。なぜパーキンソン病なのだろうか？
て幹細胞に注目が集まっている訳である。多くの研
第１の理由は、今の所パーキンソン病には満足な
究室が幹細胞、特に E S 細胞や i PS 細胞から細胞移
治療法がないためである。パーキンソン病の主な病
植治療のためのドナー細胞を作ろうとしている。こ
理像は、中脳黒質部のドーパミン神経が脱落と、そ
れらE S 細胞や i PS 細胞という多能性幹細胞は、比
の結果として神経の投射先である脳の線条体という
較的容易に増やす事が出来る。さらに最近の研究に
部分のドーパミンのレベルが下がっている状態であ
より、それらをドーパミン神経へ誘導する事が可能
る。患者さんの多くは、病気の初期には内服治療が
となった。つまり、質的および量的に安定したドー
有効である。失われた黒質−線条体系のドーパミン
パミン神経を供給する事ができる。臨床応用を考え
を補うために L −ドーパという薬剤やドーパミン作
た際、安全性等を考えると、現時点ではヒトES細
動薬が用いられる。近年では深部脳刺激療法という
胞を用いるのが最も現実味のある方法と思われる。
外科治療も一部の患者では有効である事が判ってい
しかしながら、臨床応用の前に、いくつかの解決す
る。しかし、これら２つの治療法は失われた神経回
べき問題が残っている。移植後のドーパミン神経の
路を再構築するものではない。しかも病気自体は進
不安定性、移植片の腫瘍化の危険性、動物由来の病
行してしまうため、しばしば治療を開始して10年も
原体の混入の可能性、などである。これらの問題が
経つと、症状をコントロールする事が難しくなる。
解決されれば、近い将来、細胞移植治療がパーキン
第２の理由として、中絶胎児の中脳組織を用いた
ソン病の標準的な治療に成ると期待される。
細胞移植治療が、いくつかの臨床試験で既に行われ
本講演では多能性幹細胞を用いたパーキンソン病
た事、が挙げられる。パーキンソン病に対する細胞
に対する細胞移植治療に焦点を当てる予定である。
移植治療の歴史があるので、そこで得られた知識を
幹細胞による次世代の細胞移植治療に応用する事が
57
SessionⅢ
Regenerative Medicine for Cranial Nerve Disease
Lecture 1
From bench to bed: Roadmap for clinical application
of brain repair with pluripotent stem cells.
Asuka Morizane
Postdoctoral Researcher, Dept. of Biological Repair, Field of Clinical
Application, Institute for Frontier Medical Sciences, Kyoto University
Past Records
1996 Graduated from Tokyo Medical and Dental University
2004 Ph.D., Kyoto University Graduate School of Medicine
2004 Clinical staff, Kobe City General Hospital, Department of
Neurosurgery
2004 Japanese Board of Neurosurgery
2005 Japanese Board of Neuroendovascular Therapy
2006 Postdoctoral Fellow, Section for Neuronal Survival Unit,
Wallenberg Neuroscience Center, Biomedical Center, Lund
University, Lund, Sweden
2008 Postdoctoral Researcher, Dept. of Biological Repair, Field of
Clinical Application, Institute for Frontier Medical Sciences,
Kyoto University
aborted embryos has already been performed
in several clinical trials. After several years of
systematic animal studies (1), the first clinical trial
with cell transplantation in PD was performed in
1987. Up to now, about 400 PD patients worldwide
have received this treatment. In several small
open-label trials, some grafted patients have
exhibited dramatic improvements in general
performance, increased speed of movement,
and reduction of rigidity.
18
F-fluorodopa and
11C-raclopride positron emission tomography
Introduction
(PET) scans have indicated long-lasting survival
Parkinson’
s disease (PD) is the most common
and functionality of the grafts in those patients.
movement disorder. About 1% of the population
Recently, three groups reported that transplanted
older than 60 years is affected. Although a
dopamine neurons could survive over ten years
number of brain regions are affected in PD,
after surgical postmortem examination of patients
the neuropathology that has received the most
who had undergone the procedure. However,
attention is the loss of dopamine neuron in the
Lewy body structures, pathological changes often
substantia nigra pars compacta. This leads to a
observed in the brain of PD patients, have been
decrease of dopamine in the striatum. The patients
detected in grafted cells in these long-term cases.
exhibit bradykinesia, rigidity, and tremor. The
Furthermore NIH-sponsored, double-blind, placebo-
main neuropathology in PD is loss of dopamine
controlled trails failed to observe an improvement
neurons in the substantia nigra pars compacta,
of symptoms at primary endpoints. There are
which leads to decreased dopamine level in the
several aspects that we have to consider for the
striatum. Medical treatments are useful in many
future; 1) to devise better criteria regarding patient
patients in the early stage. Patients are treated
selection ; 2) to establish the optimal numbers of
with L-3, 4-dihydroxyphenylalanine (L-dopa) and
cells to inject ; 3) to find a mild and yet effective
dopamine agonists to compensate for the loss of
mode of immunosuppression ; 4) to learn how to
dopamine neurotransmission in the nigrostriatal
avoid unwanted side-effects, such as graft induced
system. Recently deep brain stimulation, the
dyskinesias. Additionally the main problem of fetal
most common surgical procedure for PD, has
VM transplantation is the difficulty of obtaining
been reported as effective in selected patients.
the standardized quality of the donor cells from
However, these two therapies do not reconstitute
limited supply.
patients’
lost neuronal circuits and the disease itself
progresses. Thus, it is often difficult to control
Stem cells as a donor source for Parkinson’
s
the disease in late stages. Thus we do not have a
disease
satisfying treatment so far.
In contrast, stem cell research is advancing
rapidly and many people regard stem cells as
Embryonic/fetal VM transplantation
the donor source of the next generation (2). Stem
Cell transplantation using human VM of
cells are defined as undifferentiated cells that are
58
Regenerative Medicine for Cranial Nerve Disease
SessionⅢ
Lecture 1
capable of self-renewal and that can differentiate
stem cells meaning ESCs and induced pluripotent
into different cell types, i.e., they are multipotent.
stem cells (iPSCs). Those pluripotent stem cells
Three major sources of stem cells have been
can be expanded easily and recent researches
considered as donor cells for PD (Fig. 1).
enable them to differentiate into dopamine neurons
The first candidate donors for PD are the
(Fig. 2). Two main approaches are commonly used
neural stem cells (NSC), which can be derived from
to generate dopamine neurons from ESCs. One
embryonic/fetal or adult brains. This stem cell type
protocol starts by the generation of embryoid
is most probably restricted to differentiating along
bodies, from which neural progenitors are selected.
neural cell lineages, i.e., into neurons, astrocytes,
These are subjected to several defined growth
or oligodendrocytes. This type of cells is found in
factors in order to stimulate their differentiation
the developing nervous system and in the mature
into dopamine neurons (3). The other type of
brain. In human adult brain, NSCs are enriched
protocol is based on co-culturing the ESCs on
in the subventricular zone and subgranular zone
special forms of feeders (4). Regarding soluble
of the hippocampal dentate gyrus. One potential
factors that promote differentiation of ESCs into
advantage of NSCs over embryonic stem cells
dopamine neurons, specific midbrain factors, such
(ESCs) is that they are less prone to form tumors
as SHH, FGF8, and ascorbic acid, were originally
after transplantation. In general, the proportion of
shown to be able to induce the development of
NSCs that differentiates into dopamine neurons is
dopamine neurons from murine ESCs (3). A wide
low. The number of TH-immunopositive cells is
range of factors applied in different combinations
less than 2% out of the total number of cells, even
has subsequently been reported to promote
if the NSCs are derived from the rat embryonic
further the development of dopamine neurons
midbrain and are genetically modified to express
from mouse or monkey ESCs. They include IL1-
transcription factors associated with dopaminergic
β, GDNF, neurturin, TGF- β3, dibutyryl cyclic
differentiation. With regard to NSCs harvested
AMP, vitamin B12, brain-derived neurotrophic
from the adult human nervous system, the
factor, neurotrophin3, FGF20 (5), and TGF- α
generation of dopamine neurons is apparently even
. The downstream signaling pathways used by
more difficult, and to date, there is no convincing
these factors to induce a dopaminergic phenotype
demonstration that functional dopamine neurons
are not well understood. Regarding the effects of
have been obtained from such NSCs.
feeders on ESCs, several types of cells have been
The second possibility is the use of somatic
reported to have similar inducing activities. The
stem cells obtained from tissues outside the
original finding was that PA6 mouse stromal cells
central nervous system. These cell types are
promote the differentiation of mouse ESCs into
derived from specific mature tissues, such as bone
dopamine neurons (4). Subsequently, other types
marrow and skin, and from umbilical. Several
have been reported to exert similar effects, namely
well-debated studies over the past 8 years have
bone marrow stromal (MS5) cells, Sertoli cells, and
claimed that these somatic stem cells retain a
amniotic membrane. The advantage of all these co-
remarkable plasticity and can“transdifferentiate”
culture techniques is that they are simple and fast.
into cell types not normally found in the tissues in
One disadvantage is that the cellular mechanisms
which they reside. There is still no strong evidence
underlying the effects of the feeder cells are
that these stem cells can really differentiate into
completely unknown. Regarding iPSCs, we could
neurons and function as such in vivo, and certainly,
differentiate dopaminergic neurons by almost
no demonstration has shown that they can
similar method as the case of hESCs.
differentiate into dopamine neurons.
The third possibility is the use of pluripotent
59
SessionⅢ
Regenerative Medicine for Cranial Nerve Disease
Lecture 1
Basic research of transplantation using animal
around 2,100 surviving TH-immunopositive grafted
models
neurons on each side of the brain. Recently we are
The recent advancement of the differentiation
studying the effect of dopamine neurons derived
technology enables us to obtain numbers of
from human ESCs transplanted to the monkeys of
dopaminergic neurons from hESCs or hiPSCs.
Parkinson model.
Using those cells as donor for transplantation,
the effect has been examined with animal models
Problems to overcome before clinical application
of PD. So far, models of mouse, rat and monkey
Using human ES cells as a donor source for
are available. Rat model of PD is most commonly
PD seems to be the most promising strategy. Prior
used for assessment of motor behavior. The rat
to clinical application, however, several problems
is lesioned unilaterally with neurotoxin called
remain: instability of dopamine neurons in vivo ,
6-hydroxydopamine (6-OHDA). The unbalance of
risk of tumor formation by grafted cells, and the
dopamine level between sides causes rotational
possible transfer of animal-derived pathogens.
behavior to the one-side when administrated test
The survival of human ESC-derived neurons
drug such as amphetamine and apomorphine. If the
is poor following transplantation. Whereas the
grafts work properly and release dopamine in the
transplanted grafts can still contain numerous
lesioned side compensating the level of dopamine,
neurons that express a marker for neuronal nuclei
the abnormal behavior would be recovered.
(NeuN), the number of TH-immunopositive neurons
Monkey model of PD bears close resemblance to
is several-fold lower than would be expected based
human patient in many aspects. Monkeys with
on earlier intracerebral transplantation research.
lesions of the dopamine system, following 1-methyl-
Either the human ESC-derived DA neurons die
4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
during the transplantation procedure or they down-
administration, shows tremor, rigidity and akinesia.
regulate TH expression once they are grafted.
We can check the neurological motor scoring
Differences seem to exist between embryonic/
and can perform the PET scanning for functional
fetal mesencephalon-derived progenitors, which
image analysis (Fig. 3). Dopamine neurons derived
typically survive at a rate of around 10 % , and
from murine ESCs have been frequently reported
human ESC-derived progenitors/neurons. The
to survive transplantation into the striatum of
mechanisms underlying this selective vulnerability
immunosuppressed rats with a unilateral 6-OHDA
or instability of phenotype are not understood.
lesion of the nigrostriatal pathway. In some cases,
Possibly, the cells have not matured sufficiently
the grafts promote behavioral recovery in the
and can somehow“dedifferentiate”
, thus returning
recipients (6). Most of the studies have focused
to a more immature state. Or an immunoreaction
on drug-induced rotation, but some reports also
in the host environment could specifically affect
describe improved spontaneous neurological
the survival of dopamine neurons. Further studies
functions in the grafts. Primate ESCs, as well as
are needed to devise strategies to resolve this
those of mouse, can differentiate into dopamine
important issue.
neurons when co-cultured with PA6 feeder cells.
The high proliferative capacity of ESCs may
Monkey of PD have been reported to recover with
also be a disadvantage in some circumstances, as
regard to motility and posture when grafted with
they could continue to grow rapidly after being
dopamine neurons derived from primate ESCs. The
grafted into the brain. Some human ESC-derived
same monkeys also exhibit increased
F-flurodopa
grafts have been reported to form tumor-like
uptake in the striatum on positron emission
structures consisting of neuroepithelial cells, rather
tomography scans after transplantation (5, Fig. 3) ;
than teratomas that contain tissues from all germ
histological examination has revealed an average of
layers (7). The pluripotency of ESCs can pose an
60
18
Regenerative Medicine for Cranial Nerve Disease
SessionⅢ
Lecture 1
additional practical problem. It is often difficult
strategy should be encouraged because some
to exclude undifferentiated ESCs and non-neural
PD patients have shown dramatic improvement
cells completely from an ESC culture. Because
of their symptoms following embryonic/fetal
undifferentiated ESC can generate teratomas
mesencephalic transplantation. Taking all the
after transplantation, this risk also applies to
data together, we believe that stem cell therapy
intracerebral implants of ESC-derived cells. Several
will become one of the treatment options for PD
strategies have been tried to eliminate pluripotent
patients in the future.
or partially differentiated ESCs in order to avoid
tumor formation. Fluorescence-activated cell
References
sorting (FACS) has been employed in two studies
1. Brundin, P., Hagell, P. (2001) The neurobiology
describing the protocols for selecting neural
of cell transplantation in Parkison’
s disease. Clin
precursors from mixed populations including
Neurosci Res 1: 507–520
undifferentiated ESCs. Another approach to
reduce the risk of teratoma formation is to induce
2. Morizane, A., Li, J.Y., Brundin, P. (2007) From
the selective death of undifferentiated cells. For
bench to bed : the potential of stem cells for
example, the ceramide analog N-oleoyl serinol (S18)
treatment of Parkinson’
s disease. Cell Tissue
can induce apoptosis in undifferentiated cells that
Res. 331, 323-336
express Oct-4 and prostate apoptosis response-4.
The immune system is also likely to affect the
3. Lee, S.H., Lumelsky, N., Studer, L., Auerbach,
risk of tumor growth from transplanted ESCs.
Generally, allografts yield tumors more frequently
J.M., McKay, R.D., (2000)
Efficient generation of midbrain and hindbrain
than xenografts.
neurons from mouse embryonic stem cells. Nat
Typically, human ESCs are cultured on
Biotechnol 18: 675–679
mouse feeder cells with animal-derived“serum
replacements”
. Exposing human ESCs to animal
4. Kawasaki, H., Mizuseki, K., Nishikawa, S.,
derivatives leads them to incorporate N-glycolyl-
Kaneko, S., Kuwana, Y., Nakanishi, S., Nishikawa,
neuraminic acid residues (Neu5Gc) that are present
S.I., Sasai, Y. (2000) Induction of midbrain
in most mammals, except humans. Humans have
dopaminergic neurons from ES cells by stromal
circulating antibodies against Neu5Gc. Several
cell-derived inducing activity. Neuron 28: 31–40
attempts have been made to establish methods
to culture and differentiate human ESCs under
5. Takagi, Y., Takahashi, J., Saiki, H., Morizane, A.,
“feeder-free”and“xeno-free”conditions by using
Hayashi, T., Kishi, Y., Fukuda, H., Okamoto, Y.,
only human-derived products.
Koyanagi, M., Ideguchi, M., Hayashi, H., Imazato,
T., Kawasaki, H., Suemori, H., Omachi, S., Iida,
Conclusion
H., Itoh, N., Nakatsuji, N., Sasai, Y., Hashimoto,
The backbone of stem cell therapy for PD
N. (2005) Dopaminergic neurons generated
is the knowledge and experience obtained from
from monkey embryonic stem cells function
embryonic/fetal VM transplantation in the context
in a Parkinson primate model. J Clin Invest
of both basic research and clinical studies. Many
115: 102–109
problems remain to be solved before we have a
successful clinical protocol for stem cell therapy in
6. Kim, J.H., Auerbach, J.M., Rodriguez-Gomez,
PD. An important issue for clinical application will
J.A., Velasco, I., Gavin, D., Lumelsky, N.,
be the balance of benefit and safety. Nevertheless,
Lee, S.H., Nguyen, J., Sanchez-Pernaute, R.,
continued research on the cell transplantation
Bankiewicz, K., McKay, R. (2002) Dopamine
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SessionⅢ
Regenerative Medicine for Cranial Nerve Disease
Lecture 1
neurons derived from embryonic stem cells
Figure legends
function in an animal model of Parkinson’
s
Fig.1
disease. Nature 418 : 50–56
Strategies of inducing dopamine neurons from
stem cells
7. Roy, N.S., Cleren, C., Singh, S.K., Yang, L.,
There are mainly three types of stem cell sources
Beal, M.F., Goldman, S.A. (2006) Functional
that can differentiate into dopamine neurons.
engraftment of human ES cell-derived
Neural stem cells can be obtained from fetal CNS
dopaminergic neurons enriched by coculture
or subventricular zone of adult brain. Other type
with telomerase-immortalized midbrain
of the stem cells needs to“trans-differentiate”into
astrocytes. Nat Med 12 : 1259–1268
neural cells. Pluripotent stem cells meaning hESCs
or hiPSCs are the most promising ones.
Fig.2
Dopamine neurons derived from human
embryonic stem cells
The neurons were differentiated through coculture based method. The pictures are the images
of immunocytochemistry of the same fields. Those
cells are positive for the markers of tubulin β
Ⅲ (Tub β Ⅲ ; neural), tyrosine hydroxylase (TH;
dopaminergic), and Nurr1 (midbrain).
Fig.3
Dopaminergic neurons derived from monkey
ESCs surviving transplantation in the monkey
model of Parkinson’
s disease
The neurological scores improved in the group
of transplantation comparing to that of the sham
operation. PET scanning with 18F-DOPA revealed
the increased dopamine metabolism in the
transplanted group.
Fig.4
Tumor formation after transplantation in the
brain of rodents and monkeys
If there is some contamination of undifferentiated
cells in the donor population, the graft would
yield tumor after several months. Before clinical
application we have to overcome the risk of tumor
formation.
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Regenerative Medicine for Cranial Nerve Disease
SessionⅢ
Lecture 1
FIG. 1
FIG. 2
63
SessionⅢ
Regenerative Medicine for Cranial Nerve Disease
Lecture 1
FIG. 3
FIG. 4
64

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