膵外分泌細胞からの膵β細胞再生

セッションⅣ
膵β細胞の再生医療
講演２
膵外分泌細胞からの膵β細胞再生
vitro では異なるルートでもβ細胞を誘導できる可
能性があると考えるべきであろう。我々は、Cre/
loxPシステムを応用した細胞追跡法で、成体マウス
膵外分泌組織に存在する膵腺房細胞が in vitroでイ
ンスリン分泌細胞へ分化転換できることを直接的に
証明した。膵腺房細胞は膵臓で最も多量に存在する
細胞種であり、膵島移植の際にはその副産物として
大量に入手することが可能である。膵臓の細胞はす
神戸大学大学院医学研究科准教授
南幸太郎
略　歴
1990　名古屋大学大学院農学研究科博士課程前期修了
1990　修士学位（名古屋大学農学修士）授与
1990　三井製薬工業株式会社研究員
2001　千葉大学医学部助手
2002　博士学位（千葉大学医学博士）授与
2002　京都大学医学部附属病院助教授
2007　神戸大学大学院医学研究科准教授
べて共通の幹細胞から分化することから、膵腺房細
胞から移植可能なインスリン分泌細胞（膵β細胞）
を作製することは実現性が高いと思われる。しかし
ながらこれまでのところ、膵腺房細胞から誘導した
インスリン分泌細胞ではグルコースやその他の刺激
によってインスリンを分泌するものの、その能力
は実際の膵β細胞に比較すると低いという問題があ
る。実際の膵β細胞が 3 次元の構造である膵島を形
インスリンが欠乏した糖尿病の治療にはインスリ
成していることを考えると、細胞間相互作用の形成
ン注射が用いられている。しかしながら、実際の膵
が膵β細胞の機能的な最終分化に必要なのかもしれ
β細胞が血糖変化に対応してインスリンの分泌量を
ない。いずれにしても、膵腺房細胞からインスリン
細かく調整できるのに対して、インスリン注射では
分泌細胞への分化転換のメカニズムを明らかにする
血糖値を生理的範囲にコントロールすることは困難
ことが、新たに作製する細胞の質を高めることに役
であるため糖尿病によって引き起こされる様々な合
立ち、ひいては将来の糖尿病再生医療の実現のため
併症を阻止することはできない。そこで期待される
に重要であると考えている。
のが、いわゆる幹/前駆細胞からインスリン分泌細
胞を誘導して移植治療する再生医療である。胚性
幹（ES）細胞からは実際にインスリン分泌細胞を
誘導できることが報告されている。しかし、ES細
胞の使用は臨床応用の面でいくつかの障害を抱えて
いる。たとえば、E S 細胞由来の細胞を移植すると
腫瘍が形成される可能性があるし、ヒトのE S 細胞
の取得には倫理的問題もある。加えて、E S 細胞か
ら作製したインスリン分泌細胞は正常の膵β細胞と
は異なり、インスリンの産生・分泌が異常である可
能性も指摘されている。昨今話題の i P S 細胞につい
ても倫理的問題を除けば、インスリン分泌細胞の作
製においてES 細胞と同様の問題が残る。一方、膵
導管細胞や oval 細胞、骨髄細胞などの成体に存在す
る非β細胞からインスリン分泌細胞を誘導できると
の報告もある。成体の膵β細胞はβ細胞自体の複製
によってのみ維持されるとの報告がなされたが、in
88
Regenerative Medicine for Pancreatic Beta Cells
SessionⅣ
Lecture 2
Regeneration of pancreatic β- cells from pancreatic
exocrine cells
Kohtaro Minami
acinar cells can readily be obtained as a byproduct
Associate Professor, Kobe University Graduate School of Medicine
of islet transplantation. Considering that all types
Past Records
1990 Completed Master Course, Nagoya University Graduate
School of Agricultural Science
1990 Received M.S. (Agricultural Science), Nagoya University
1990 Scientist, Mitsui Pharmaceutical Industry
2001 Assistant Professor, School of Medicine, Chiba University
2002 Received Ph.D. (Medical Science), Chiba University
2002 Associate Professor, Kyoto University Hospital
2007 Associate Professor, Kobe University Graduate School of
Medicine
of pancreatic cells originate from the same stem
Insulin has been used in the treatment of
Although cells in the pancreas seem to be
diabetes with absolute insulin deficiency since its
static, in certain situations including pathological
discovery. However, normal pancreatic β-cells
conditions and experimental models, remodeling
continually adjust insulin secretion in response
of the pancreas occurs.
to varying blood glucose levels, while exogenous
pancreatic duct in rats, replacement of exocrine
insulin administration cannot maintain blood
acini by duct-like structures is observed (18). This
glucose levels within the narrow physiological
acinoductal metaplasia has been thought to be at
range that protects from development of
least in part due to transdifferentiation of amylase-
the various diabetic complications.
Thus,
positive pancreatic acinar cells into amylase-
transplantation of insulin-secreting cells generated
negative and cytokeratin-positive duct-like cells
from stem/progenitor cells is a promising option
(19). By treating the rats with dexamethason to
for the treatment of insulin-deficient diabetes (1-
inhibit loss of amylase expression, transitional cells
3). Although it has been reported that embryonic
co-expressing amylase and cytokeratin 20 were
stem (ES) cells can be manipulated to produce
detected (19), supporting the notion of acinar-to-
insulin (4-7), there are several obstacles to their
ductal transdifferentiation.
clinical use. Transplantation of ES cell derivatives
positive cells that also express amylase were found,
into human recipients can result in the formation
indicating acinar-to-endocrine transdifferentiation.
cells (17), the generation of transplantable insulinsecretingβ-cells from pancreatic acinar cells would
seem to be feasible.
Regeneration in the pancreas
After ligation of the
Moreover, insulin-
of ES cell-derived tumors (8). Ethical problems
arise in the acquisition of human ES cells (9). In
Stem/progenitor cells in the pancreas
addition, it has been suggested that both the
Many attempts have been made to identify
production and the release of insulin in such
stem/progenitor cells in adult pancreas. Zulewski
manipulated ES cells may be abnormal (10,11).
et al . showed that cells expressing the neural
Generation of β-cells from induced-pluripotent
stem cell marker nestin occur in human and
stem (iPS) cells has similar problems to those in
rat pancreatic islets, and that these cells can be
ES cells except ethical ones. On the other hand,
isolated and cultured for a long time (20). It has
recent studies have shown that insulin-secreting
been shown that cultured nestin-positive cells
cells can be generated in vitro from adult non-
can be differentiated into insulin-producing cells
β-cells including mouse and human pancreatic
(20,21), and that such cells from human fetal
duct cells (12-14), rat hepatic oval cells (15), and
pancreas when transplanted can be expanded and
mouse bone marrow cells (16). Pancreatic acinar
differentiated into islet-like cell clusters, which
cells represent the most abundant cell type in the
can reverse hyperglycemia in diabetic mice (22).
pancreas. In addition, a large number of pancreatic
Clonal identification of multipotent precursors from
89
SessionⅣ
Regenerative Medicine for Pancreatic Beta Cells
Lecture 2
adult mouse pancreas has recently been reported
Generation of beta-cells from pancreatic acinar
(23). These candidate progenitor cells proliferate
cells
in the serum-free conditions used for neural stem
In vitro generation of insulin-secreting cells
cell (NSC) culture, and form spherical cell clusters
(beta-cells) from pancreatic acinar cells has been
like NSCs by floating culture.
The spheroids
reported by several groups (31,33,34). Since a
containing progenitor cells can be derived from
large number of pancreatic exocrine cells can be
both pancreatic islets and duct cell populations.
obtained as a byproduct of islet transplantation, the
Although the cells were generated from both
exocrine pancreas is an intriguing source for the
nestin-positive and nestin-negative fractions, they
generation of transplantable surrogate beta-cells.
all expressed nestin during the expansion period.
Song et al . found that adult rat pancreatic acinar
Interestingly, the cells in the spherical clusters
cells transdifferentiated into insulin-expressing cells
show characteristics of both pancreatic and neural
in vitro (33). They isolated pancreatic acinar cells
precursors. Considered together, these findings
from adult rats and cultured the cells in suspension
suggest that while stem/progenitor-like cells can
without adding any growth factor and cytokines.
be obtained from adult pancreas, it is not yet clear
Most of the acinar-derived cells lost amylase
that such isolated stem/progenitor-like cells have
expression and acquired a ductal phenotype within
full potential to differentiate into native pancreatic
a week. Insulin-positive cells were detected at the
beta-cells and function as stem/progenitors in the
peripheral of the spherical cell clusters derived
pancreas in vivo .
from the acinar cells (33).
Baeyens et al . also
have shown that rat exocrine pancreatic cells can
Transdifferentiation in the pancreas
transdifferentiate into insulin-secreting cells by
Although it has been thought that terminally
cultivation in the presence of EGF and LIF (34).
differentiated cells do not change their phenotype,
However, neither direct evidence of the origin
accumulating evidence suggests that phenotypic
of these cells nor their precise insulin secretory
plasticity is retained in differentiated cells.
properties was shown in these studies.
Transdifferentiation, the conversion of one already
We have established a method for the
differentiated cell type to another, is a paradigm
generation of insulin-secreting cells from
of phenotypic plasticity in adult cells. In general,
pancreatic acinar cells in mice (31). The pancreatic
such phenotypic change occurs in tissues with
exocrine cell-enriched fraction was prepared by
chronic damage and in tissue regeneration (24).
Ficoll density gradient centrifugation. By this
The pancreas is an organ in which metaplasia, a
method, pancreatic exocrine cells are recovered
pathological state involving transdifferentiation
as a pellet, and are then stained with dithizone,
(25,26), frequently occurs.
For example,
a zinc-chelating agent, to remove contaminated
hepatocyte-like cells appear in human pancreatic
pre-existing pancreatic beta-cells. The resulting
cancer in some cases (27), and experimental
fraction contains >90 % of amylase-positive cells,
conditions such as copper depletion can lead to the
approximately 5 % of cytokeratin-positive cells,
development of pancreatic hepatocytes in rodents
and less than 0.01% of insulin-positive cells. The
(28,29).
Moreover, metaplastic hepatocytes in
exocrine cell-enriched fractions were then
ciprofibrate-treated rat pancreas have been shown
cultured in RPMI-1640 medium supplemented
to originate from pancreatic exocrine acinar cells
with 0.5% fetal calf serum and 20 ng/ml of EGF.
(30). Pancreatic acinar cells also can convert into
Under these conditions, the cells readily formed
insulin-secreting cells, and direct evidence for
aggregates and became smooth spheroids within
acinar-to-endocirne transdifferentiation has been
a few days. When sticky cell culture dishes were
provided both in vitro (31) and in vivo (32).
used, the cells began to adhere, and formed small
90
Regenerative Medicine for Pancreatic Beta Cells
SessionⅣ
Lecture 2
monolayer colonies.
We found that a subset
cells, and cultured as described. After the culture,
of the cells in these colonies expressed insulin.
ECFP-expressing insulin-positive cells were
Most insulin-positive cells were detected at the
frequently found, demonstrating that the insulin-
peripheral of small colonies.
positive cells originate from amylase-expressing
However, insulin
secretion was undetectable under these conditions.
pancreatic acinar cells (Figure 2b) (20).
When the isolated pancreatic exocrine cells were
The most important function of pancreatic
cultured in suspension, insulin production was
beta-cells is glucose-induced insulin secretion, in
increased compared to that in monolayer culture.
which several key molecules are known to be
Pancreatic exocrine-derived spheroids could be
involved (36). Glucose transporters, glucokinase,
maintained throughout the culture (Figure 1). The
ATP-sensitive K + (KATP) channels, voltage-
frequency of insulin-positive cells was increased
dependent Ca 2+ channels, molecules associated
to 〜5% of total cells on day 4. The insulin-positive
with the exocytotic machinery (SNAREs), and
cells also expressed C-peptide, indicating de
prohormone convertases (PC1/3 and PC2) are
novo biosynthesis of insulin in these cells. To
required for proper glucose-induced insulin
determine the cell type that differentiates into
secretion. The expressions of all of these molecules
insulin-producing cells in exocrine pancreas,
were induced or up-regulated in pancreatic acinar
we first evaluated the expression of pancreatic
cells after the culture. The expression profiles of
cell markers during culture. Both amylase and
the exocrine pancreas-derived insulin-producing
elastase, which are acinar cell-specific enzymes,
cells became similar to those of the pancreatic
were strongly detected before culture (day 0) at
islets. In addition, formation of insulin-containing
both mRNA and protein levels. However, their
granules was confirmed by immunoelectron
expressions were drastically decreased during
microscopy, indicating that these cells can secrete
culture, becoming barely detectable on day 4 and
insulin in a regulated manner.
after. Immunocytochemistry showed that amylase-
concentration of KCl stimulated insulin secretion
positive cells comprised more than 90 % of total
from the cells, indicating the occurrence of Ca2+-
cells in the initial preparation (day 0), but less than
triggered exocytosis in pancreatic acinar-derived
5% 4 days after culture. Although insulin-positive
cells.
cells were generally negative for amylase, a few
glibenclamide, a sulfonylurea widely used in
cells clearly expressed both insulin and amylase.
treatment of diabetes, indicating that functional
These results suggest that the insulin-producing
K ATP channels are expressed.
Indeed, a high
Insulin secretion also was increased by
Importantly,
cells in the culture are derived from amylase-
glucose stimulated insulin secretion from exocrine
expressing mature pancreatic acinar cells.
pancreas-derived cells in a concentration-
To confirm that the newly-made insulin-
dependent manner, demonstrating that the cells
producing cells were derived from pancreatic
are glucose responsive. In addition, GLP-1 (7-36
acinar cells, we utilized the method of cell lineage
amide), an incretin, potentiated insulin secretion
tracing. In this method, ROSA26-loxP-stop-loxP-
in the presence of relatively high concentrations
ECFP reporter mouse (R26R-ECFP) expressing
of glucose, indicating that the cAMP-mediated
enhanced cyan fluorescent protein (ECFP) that can
potentiation system also is present in the cells
be activated through the action of Cre recombinase
(Figure 3). These results show that pancreatic
(35) and adenoviruses in which either amylase-2
acinar-derived cells have qualitatively similar
or elastase-1 promoter drives the expression of
insulin secretory properties to those of native
Cre recombinase are used (Figure 2a). Pancreatic
pancreatic beta-cells. However, insulin content
exocrine cells from R26R-ECFP mice were infected
in these pancreatic acinar-derived cells (including
with these adenoviruses to label pancreatic acinar
both insulin-positive and insulin-negative cells)
91
SessionⅣ
Regenerative Medicine for Pancreatic Beta Cells
Lecture 2
is only 1/400 that of native pancreatic islets.
inhibitor (Figure 4).
Since the frequency of insulin-positive cells is
approximately 5% of total cells in the culture, the
Perspectives
insulin content of the newly made insulin-positive
Generation of pancreatic β -cells from non-
cells is about 1/20 that of a native beta-cell (20).
β-cells in vitro represents a potentially useful
We found that enzymatic dissociation of the
approach to cell replacement therapy for type
pancreas leads to activation of the EGF receptor
1 diabetes. Accumulating evidence shows that
and its downstream signaling. The EGF receptor
insulin-secreting cells can be generated from
was not activated before dissociation but was
pancreatic acinar cells by transdifferentiation in
activated (tyrosine phosphrylated) by enzymatic
vitro under certain culture conditions. However,
dissociation. When an inhibitor of EGF receptor
such newly made cells are not fully differentiated
kinase (AG1478) was applied, transdifferentiation
beta-cells, as assessed by both insulin secretory
of isolated pancreatic acinar cells into insulin-
properties and gene expression profile, compared
secreting cells was inhibited (31), demonstrating
with native pancreatic beta-cells. Native pancreatic
that activation of the EGF receptor is essential for
beta-cells are highly differentiated cells equipped
the transdifferentiation. In embryonic pancreas,
with a well-regulated secretory apparatus of insulin
EGF increases the number of undifferentiated
secretion that controls blood glucose levels within
endocrine precursor cells, and, upon removal of
a narrow physiological range. Considering that
EGF, a large number of beta-cells are differentiated
the native pancreatic beta-cells form the three-
(37), suggesting that EGF may be important for
dimensional structure (islet) with other types of
the proliferation of endocrine precursors and/or
endocrine cells, intercellular communication might
endow the cells with commitment to endocrine
be an important factor for full differentiation into
lineage. In addition, in TGF-alpha transgenic mice,
mature beta-cells from non-beta-cells. In addition,
ductal hyperplasia and pronounced interstitial
cell-to-extracellular matrix contact could also be
fibrosis occur in exocrine pancreas (38). In these
implicated in the establishment of cell functions.
mice, numerous duct cells of the pancreas have
Therefore, molecules associated with cell-to-cell
both zymogen and mucin granules (38).
Islet
and cell-to-matrix interactions may have roles in
neogenesis from the metaplastic duct is observed
the transdifferentiation. In any case, we need to
in the pancreas of these transgenic mice (39).
pay more attention to the morphological aspect of
TGF-alpha is a member of the EGF family acting
newly-made insulin-secreting cells for the use of
via the EGF receptor. Thus, EGF signaling is also
cell transplantation. Clarification of the molecular
involved in acinar-to-ductal transdifferentiation
mechanisms of such transdifferentiation as well as
and islet neogenesis in adult pancreas. In addition,
the acquisition of insulin secretory function in the
we have found that formation of spherical three-
process may provide a basis for cell replacement
dimensional structures by cadherin-mediated cell-
therapy in type 1 diabetes.
cell adhesion plays a critical role in the induction
of β-cell-specific gene expression in acinar-derived
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Lecture 2
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94
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39. Gu, D., and Sarvetnick, N. (1993) Epithelial cell
Regenerative Medicine for Pancreatic Beta Cells
SessionⅣ
Lecture 2
proliferation and islet neogenesis in IFN-g
Figure 4. Model of in vitro transdifferentiation of
transgenic mice. Development 118, 33-46.
pancreatic acinar cells. Enzymatic dissociation of
exocrine pancreas disrupts epithelial structures
Figure legends
of acini, resulting in the loss of cadherin-mediated
Figure 1. Pancreatic acinar cell-derived spherical
cell-cell adhesion, which causes dedifferentiation
cell clusters. Phase contrast photomicrographs
of the acinar cells. Meanwhile, EGF receptors
of cultured pancreatic acinar cells.
Isolated
are activated, followed by activation of the PI3-
pancreatic acinar cells were cultured in suspension
kinase pathway. Then, cadherin-mediated cell-cell
in the presence of EGF.
adhesion is recovered by the enhanced expression
The cells began to
aggregate within a few days and formed smooth
of E-cadherin and β-catenin.
spheroids after 4 days of culture.
essential for redifferentiation of the dedifferentiated
This recovery is
cells into insulin-secreting cells.
Figure 2. Cell lineage tracing by Cre/loxP-based
system. (a) The scheme of pancreatic acinar cell
specific cell marking. In cells from the R26RECFP mouse, expression of the fluorescent protein
(ECFP) is activated through the action of Cre
recombinase to remove a transcriptional“stop”
sequence.
When amylase/elastase-expressing
acinar cells are infected with adenovirus
expressing Cre recombinase under control of
either amylase or elastase promoter, the cells
are labeled permanently with ECFP. (b) Lineage
tracing of labeled acinar cells. Pancreatic acinar
cells from R26R-ECFP were labeled by infection
of Ad-pAmy-Cre at approximately 50 % efficiency.
Because fluorescence of ECFP is diminished after
fixation, ECFP-expression was detected using antiGFP antibody. Cells positive for insulin (arrow
heads), ECFP (arrows), and both insulin and
ECFP (asterisks) are observed. Scale bar, 20 μm.
Reprinted from Ref. 31 with modification, copyright
(2005) National Academy of Science, U.S.A.
Figure 3. Insulin secretion was stimulated by
30 mM KCl, 0.1μM glibenclamide (Glib), 0.1mM
carbachol (CCh), or increased concentrations of
glucose (G3 ; 3 mM, G10; 10 mM, G20 ; 20 mM) for
60 min. Potentiation by GLP-1 (7-36 amide) (100
nM) is also shown. Data are means ± S.E. of three
to seven independent experiments.
Reprinted
from Ref. 31 with modification, copyright (2005)
National Academy of Science, U.S.A.
95
SessionⅣ
Regenerative Medicine for Pancreatic Beta Cells
Lecture 2
Figure 1
Figure 2
96
Regenerative Medicine for Pancreatic Beta Cells
SessionⅣ
Lecture 2
Figure 3
Figure 4
97

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