転写因子を用いた膵β細胞の再生

膵β細胞の再生医療
講演１
セッションⅣ
転写因子を用いた膵β細胞の再生
⒝ 組織幹細胞　我々はマウス膵から膵内分泌細
胞、外分泌細胞、アルブミン陽性細胞に分化しうる
膵由来組織幹細胞の単離培養に成功した。この細胞
は膵管特異的遺伝子を発現し、pdx-1が陽性で増殖
力がある。我々はこの幹細胞の細胞分化に対する転
写因子遺伝子の影響を効率よく検討するために、エ
ピゾーマルに外来遺伝子を維持し安定した外来遺伝
子発現が可能な細胞系の確立を試み、その結果、種々
大阪大学大学院医学系研究科幹細胞制御学准教授
倭　英司
の遺伝子の組み合わせで膵外分泌細胞や特定の膵内
分泌細胞に分化誘導可能であることを確認してい
1984　大阪大学医学部卒業
1990
医学博士（大阪大学）
1991
米国ハーバード大学ジョスリン糖尿病研究所研究員
1998
大阪大学大学院医学系研究科幹細胞制御学准教授
1991−1993 若年糖尿病財団特別奨学金
1992
米国臨床研究連盟ヘンリー・クリスチャン優秀研究
賞
2001
日本医師会医学研究助成
る。また、この系を利用してインスリン遺伝子発現
転写因子は細胞分化や臓器発生に必須であり、膵
た膵への遺伝子導入法を開発した。この方法を用い
発生や膵ベータ細胞分化に関与する転写因子も報告
てPdx-1遺伝子およびIsl-1遺伝子を導入することに
されている。そこで、我々の研究室では幹細胞や生
より、膵内に ductal complex 形成を誘導し、イン
体内への遺伝子導入方法の開発とそれを利用した転
スリン産生細胞も再生可能であることを示してき
写因子遺伝子導入により、幹細胞の分化誘導や分化
た。さらに近年、Pdx-1もしくはIsl-1遺伝子、ある
細胞の分化転換を用いた膵ベータ細胞再生を試みて
いは両遺伝子を部位特異的に薬剤誘導可能なマウス
いる。
を作成した。現在、このマウスを用いて、膵島再生
略　歴
受賞歴
のマーカー遺伝子として truncated CD4を用いる方
法も考案し検討中である。
２　in vivoにおける検討
我々は膵管経由でアデノウイルスベクターを用い
およびそれらの再生細胞の細胞系譜に関する検討を
１　in vitroにおける検討
行っており、転写因子遺伝子の細胞転換に関するin
⒜ ES 細胞　近年、E S 細胞様の未分化細胞 i PS
vivo における役割を明らかにしようとしている。
細胞を体細胞から樹立され、E S 細胞樹立に伴う倫
理的な問題の回避が可能となった。E S 細胞を用い
た分化誘導法の確立はiPS細胞にも応用が可能であ
り、さらなる検討が急務である。我々は外来遺伝子
の薬剤誘導ユニットをE S 細胞のROSA26 locusに組
み込むことにより、細胞の分化状態に関わらず遺伝
子をon-off できる系を確立した。この細胞を用い、
Pdx-1遺伝子導入によりインスリン産生細胞の分化
誘導効率が向上することや Sox17 遺伝子導入により
胚胎外内胚葉細胞を分化誘導することに成功した。
またES 細胞由来胚胎外内胚葉細胞にさらにアデノ
ウイルスベクターを用いた転写因子遺伝子導入によ
り、インスリン産生細胞を分化誘導が可能であるこ
とを示している。
81
SessionⅣ
Regenerative Medicine for Pancreatic Beta Cells
Lecture 1
Regeneration of pancreatic β cells by means of
transcription factor genes
Eiji Yamato
vector was stably transfected. Using these cells,
Associate Professor, Division of Stem Cell Regulation Research,
Osaka University Graduate School of Medicine
Past Records
M.D., Osaka University Medical School
1984
Ph.D., Osaka University Medical School
1990
Research Fellow, Joslin Diabetes Center, Boston, USA
1991
Associate Professor, Division of Stem Cell Regulation
1998
Research, Osaka University Graduate School of Medicine
Special Awards
1991−1993 Juvenile Diabetes Foundation Postdoctoral Fellowship
Award
The Henry Christian Award for Excellence in Research
1992
from the American Federation for Clinical Research
Research Support Award of the Japan Medical Association
2001
we can transfect the multiple gene-expression
vectors effectively. As a result, the coexpression
of Mafa , Neurod1 , and Ipf1 induced Ins1 and
Ins2 expression in PLT-PPPD cells. The forced
expression of Pax6 alone induced the expression
of glucagon. The coexpression of Neurod1 and
Isl1 induced Ins2 and Sst expression . On the
other hand, the expression of Ptf1a and Foxa2
induced the expression of exocrine markers
Summary
Cpa1 and Amy2 . Transfections with multiple
Cell replacement therapy is a desirable
transcription factors showed that Isl1 is required
option for the restoration of tissue function.
for the differentiation of both insulin-positive cells
ES cells derived from early embryos might
and somatostatin-positive cells. In addition, Foxa2
represent a limitless source of specific cell types
induced the differentiation of glucagon-positive
for transplantation.
cells and inhibited the differentiation of insulin-
Transcription factors, in
combination with environmental factors, are
positive and somatostatin-positive cells.
crucial for the differentiation of organs. To derive
We have also examined the effect of gene
a restricted cell lineage from ES cells, we have
transfer of transcription factors in vivo. Among
established an ES cell line in which expression
various transcription factors, we showed that
of Ipf1 gene, which have roles in differentiation,
adenovirus expressing Isl1 as well as Ipf1 ,
were regulated using drug.
And we showed
administered via common bile duct, can induce
forced expression of Ipf1 in ES cells, which were
the beta cell neogenesis and ductal proliferation
initiated the differentiation by embryoid body,
in the pancreas. This gene transfer method was
produced not only insulin gene but also insulin
also analyzed and was revealed that the cells
protein. We further examined the ES cell derived-
which expressed the ectopic gene were almost
endodermal cells, which were obtained by the
exocrine cells. Thus exocrine cells seemed to
forced expression of Sox17 gene and showed the
be transdifferentiated to CK-positive cells which
combination of Ipf1 , Neurod1 , and Mafa genes
formed the duct-like structure and several cells in
were efficient for the induction of insulin gene.
the duct-like structure may form the new islets.
Pancreatic stem cells are another source for
regeneration of insulin-producing cells. We have
Introduction
succeeded to obtain the duct-like pancreatic stem
cells (PPPD cells). Our method can selectively
As an individual insulin-producing cell is
isolate the pdx-1-positive stem cells. Cell clone
functional, they are good target of regenerative cell
can obtained from these stem and they can
therapy. There are two sources for the material
differentiate into insulin-producing cells, amylase-
for cell therapy, differentiated cells and stem cells.
producing cells, or albumin-producing cells. We
Although differentiated cells are already functional,
have recently established the PPPD cells in
they do not have the capacity for proliferation, so it
which polyoma large T (PLT)-expressing plasmid
is difficult to obtain enough cell number for cure of
82
Regenerative Medicine for Pancreatic Beta Cells
SessionⅣ
Lecture 1
diabetes and it is requisite to immortalize the cells
Recently, it was proven that Yamanaka were
to proliferate the differentiated cells. In contrast,
able to establish an iPS cell similar to the ES cell
stem cells are less problematic for proliferation,
with the pluripotency by introducing four genes
but stem cells should be differentiated to functional
(Oct3, Sox2, Klf4, and c-myc) into the fibroblast.
insulin-producing cells to serve as a source for
The strategy of differentiation accumulated by
therapy.
the ES cell will be useful for this somatic origin
Stem cells are consisted from 2 groups,
pluripotent stem cell without considering the
embryonic stem (ES) cells and adult stem cells. ES
ethical problem that the ES cell has possessed.
cells have a capacity to differentiate into all the
cells which consists the body. Recent study also
revealed that adult stem cells also have a capacity
to differentiate into various cells under specific
2. ES cells and insulin-producing cells
(1) The induction of differentiation of ES cells by
transcription factor genes
circumstances. Thus strategy for differentiation
The transcription factor gens are thought to
of stem cells into a specific lineage will contribute
be indispensable to the differentiation progress, and
the progression of cell therapy for regenerative
the differentiation of the pancreatic beta cell was
medicine.
also provided by various transcription factors as
Alternative approach for regenerative
for. Thus, we established the ES cells in which the
medicine is a strategy for an induction of
induced expression of the foreign gene is possible
endogenous stem cells to a certain differentiated
by the drug. Using this technique, it turns out to
cells or an induction of transdifferentiation of the
obtain the cell of the extraembryonic endoderm
differentiated cells. In this review, the trial of
cells by the forced expression of GATA4 and the
regeneration of insulin-producing cells both in vitro
GATA6 gene, among various endodermal-specific
and in vivo a special concern with differentiation
genes which are essential for promotion of the
and regeneration from the stem cells (Figure 1).
differentiation [1].
It is known that the transgene was
Insulin-producing cells differentiated from ES cells
gradually suppressed in its expression when it
1. ES cells and regenerative medicine
is introduced into ES cell. Then, we established
The ES cell is a cell established from the
the ES cell line in which drug-inducible gene-
internal cell mass of the blastocyst, and has the
expressing unit was inserted onto the ROSA26
pluripotency. The nucleus of the somatic cells is
gene locus, their expression was continuous
reprogrammed when the nucleus of somatic cells
during the differentiation and made the ES cell
is transplanted to the enucleolated unfertilized
line that the genetic control is possible even after
egg, and the embryo with the genetic information
the differentiation. As a result, ES cells can be
derived from the somatic nucleus origin is
differentiated into insulin producing cells highly
generated. Making the embryo with the genetic
effectively when pdx-1 gene is introduced during
code of each person's somatic nucleus from this
differentiation [2]. Moreover, these cells decrease
technically became possible (cloned embryo).
their expression level of insulin by long-culture,
As the embryonic stem cell is established from
but the insulin gene level was recovered by
the internal cell mass of the blastocyst stage of
introducing Pdx-1 and Beta2 genes [3].
this cloned embryo, the functioning cell will be
differentiated from the self-somatic nucleus origin
(2) Differentiation of insulin-producing cells from
embryonic stem cell, and cell transplants without
the endodermal cells derived from ES cells
the rejection become possible (therapeutic cloning,
(Figure 3)
Figure 2).
We have reported that the insulin gene
83
SessionⅣ
Regenerative Medicine for Pancreatic Beta Cells
Lecture 1
appeared even if the embryonic stem cell that
ductal cells exists is thought. For instance, an
knocked out the pdx-1 gene and that pdx-1,
increase of the new insulin-positive cell in the duct
which is requisite for the proper differentiation
and the pdx-1 positive duct cells were observed
of pancreatic beta cells was dispensable in the
in pancreatic injury model (90 % pancreatectomy
differentiation of insulin-producing cells from the
and pancreatic duct ligature model) in the adult
ES cells and showed the possibility that these
rat and the pancreatitis model and certain kinds
insulin-producing cells were not derived from
of transgenic mice. Thus it is assumed that the
the definitive endoderm [4]. Moreover, we have
pdx-1 positive duct cell which has the proliferation
examined the insulin-producing cells from ES
capacity is pancreatic stem cell.
cells, in which the marker gene was expressed
under the insulin promoter and revealed that the
insulin-producing cells obtained from a previous
2. dedifferentiation and transdifferentiation of
pancreatic exocrine cells
differentiation method were originated from the
There is a report that it was possible to
primitive endoderm [5].
differentiate into the ductal cell from exocrine cells
Then, we have tried to improve the strategy
in vitro. Moreover, in the transgenic mouse in
for the differentiation of insulin-producing cells
which TGFα or amphiregulin gene was ectopically
from the endodermal cells derived from ES cells.
expressed in exocrine cells, the massive duct-like
We succeeded to obtain extraembryonic endoderm
structure was observed and pdx-1 positivity cell
cells by floating culture with high efficiency by
in the ductal cells in TGF α transgenic mouse.
ectopic expression of sox17 gene under the LIF
Thus differentiation from exocrine cells into the
existence. In addition, when the gene necessary
insulin producing cells via the ductal cell could be
for the beta cell of pancreas differentiation was
achieved.
introduced into the ES-derived extraembryonic
It is known that to the AR42J cells of the
endodermal cells, the insulin-producing cells were
rat acinar cell origin, which express various
obtained (manuscript in preparation). As when
transcription factor genes indispensable for the
these genes are introduced into the ES cell, they
pancreatic development, differentiate into the
did not induce the insulin gene expression, the
insulin producing cells by adding various growth
extraembryonic endodermal cells derived form
factors. Recently, Minami et al. reported that
ES cells can become the material of the insulin-
they isolate the exocrine cell by using cell-lineage
producing cell.
tracing, and showed that it had found the cell could
be differentiated into the insulin producing cell.
Insulin production life cell reproduction that
We succeeded in the isolation culture of the
uses pancreatic cell
pancreatic duct-like cells from the adult mice.
It is thought that the pancreatic stem cell is
This cell contains a lot of amylase positivity cells
one of the tissue stem cells, and they differentiated
at first, and has the possibility that they are the
into the needed cell when reproduction stimulation
exocrine cell origin.
was added (Figure 4). The regenerative medicine of
high proliferating ability by devising the culture
the diabetes can be also achieved by reproducing
condition, possible the long-term culture of one
the insulin-producing cells using pancreatic stem
year or more even by serum-free media, and
cell.
differentiate into pancreatic endocrine and exocrine
This cell can maintain a
cells, and also into albumin-producing cells by
1. Pancreatic duct cell-origin stem cell
the change of the culture condition and the gene
The possibility where the cell that functions
transfer [6].
as a pancreatic stem cell partially of pancreatic
84
Regenerative Medicine for Pancreatic Beta Cells
SessionⅣ
Lecture 1
Conclusion
The insulin producing cell is clarified to be
reproduced even though it is insufficient yet. It
is thought that promotion of the research of the
regenerative medicine of the diabetic in the future
is indispensable for a lot of diabetics who hope the
diabetic cures.
References
1. Fujikura, J., Yamato, E., Yonemura, S., Hosoda,
K., Masui, K., Nakao, K., Miyazaki, J-I., Niwa, H.
(2002) Differentiation of embryonic stem cells is
induced by GATA factors. Genes Dev 16, 784789.
2. Miyazaki, S., Yamato, E., Miyazaki, J. (2004)
Regulated expression of pdx-1 promotes in vitro
differentiation of insulin-producing cells from
embryonic stem cells. Diabetes 53 : 1030-1037.
3. Saitoh, K., Yamato, E., Miyazaki, S., Miyazaki,
J. (2007) Both Pdx-1 and NeuroD1 genes are
requisite for the maintenance of insulin gene
expression in ES-derived differentiated cells.
Diabetes Res Clin Pract 77 Suppl 1 : S138-S142.
4. Takayama, I., Miyazaki, S., Tashiro, F., Fujikura,
J., Miyazaki, J., Yamato, E. (2008) Pdx-1independent differentiation of mouse embryonic
stem cells into insulin-expressing cells. Diabetes
Res Clin Pract 79 : e8-10.
5. Moritoh, Y., Yamato, E., Yasui, Y., Miyazaki, S.,
Miyazaki, J. (2003) Analysis of insulin-producing
cells during in vitro differentiation from feederfree embryonic stem cells. Diabetes 52 : 11631168.
6. Yamamoto, T., Yamato, E., Taniguchi, H. Shimoda,
M., Tashiro, F., Hosoi, M., Sato, T., Fujii, S.,
Miyazaki, JI. (2006) Stimulation of cAMP
signalling allows isolation of clonal pancreatic
precursor cells from adult mouse pancreas.
Diabetologia 49 : 2359-2367.
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Regenerative Medicine for Pancreatic Beta Cells
Lecture 1
Regenerative Medicine for Pancreatic Beta Cells
SessionⅣ
Lecture 1
87

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