DEVELOPMENT AND STEM CELLS
RESEARCH ARTICLE 2289
Development 137, 2289-2296 (2010) doi:10.1242/dev.048421
© 2010. Published by The Company of Biologists Ltd
Adult pancreatic acinar cells give rise to ducts but not
endocrine cells in response to growth factor signaling
Stacy A. Blaine1, Kevin C. Ray1, Reginald Anunobi1, Maureen A. Gannon2,3,4, Mary K. Washington5 and
Anna L. Means1,3,*
SUMMARY
Studies in both humans and rodents have found that insulin+ cells appear within or near ducts of the adult pancreas, particularly
following damage or disease, suggesting that these insulin+ cells arise de novo from ductal epithelium. We have found that
insulin+ cells are continuous with duct cells in the epithelium that makes up the hyperplastic ducts of both chronic pancreatitis
and pancreatic cancer in humans. Therefore, we tested the hypothesis that both hyperplastic ductal cells and their associated
insulin+ cells arise from the same cell of origin. Using a mouse model that develops insulin+ cell-containing hyperplastic ducts in
response to the growth factor TGF, we performed genetic lineage tracing experiments to determine which cells gave rise to
both hyperplastic ductal cells and duct-associated insulin+ cells. We found that hyperplastic ductal cells arose largely from acinar
cells that changed their cell fate, or transdifferentiated, into ductal cells. However, insulin+ cells adjacent to acinar-derived ductal
cells arose from pre-existing insulin+ cells, suggesting that islet endocrine cells can intercalate into hyperplastic ducts as they
develop. We conclude that apparent pancreatic plasticity can result both from the ability of acinar cells to change fate and of
endocrine cells to reorganize in association with duct structures.
INTRODUCTION
Traditionally, terminally differentiated cells of mammals were
thought to be immutable, that is, confined to their final
differentiated state for the remainder of their lifespans. However,
in cases of injury or disease, the cellular composition of organs
has been noted to change. For example, in both chronic
pancreatitis and pancreatic cancer, many of the enzymeproducing acinar cells in the affected region of the pancreas are
absent and replaced by hyperplastic ductal epithelium (Kloppel,
1993; Kloppel and Maillet, 1998). In this and other similar cases,
it has been unclear how much of the change in composition
results from reprogramming of mature cells and how much from
stimulation of progenitor cells residing either within the organ or
in the bone marrow. Complicating the issue of origin, small
clusters of endocrine cells, notably insulin-producing cells, are
closely associated with the hyperplastic ducts that arise during
chronic pancreatitis and pancreatic cancer (Chen et al., 1988;
Esposito et al., 2007; Phillips et al., 2007). This association
between duct and insulin-expressing (insulin+) cell reflects the
relationship between these tissues in normal embryonic
development, when insulin-producing  cells differentiate from
progenitors within the duct-like epithelium of the developing
pancreas. Thus, the presence of insulin+ cells in hyperplastic
ducts suggests that the adult pancreas is capable of recapitulating
embryonic development for the production of new insulin-
1
Department of Surgery, 2Department of Medicine, 3Department of Cell and
Developmental Biology, 4Department of Molecular Physiology and Biophysics, and
5
Department of Pathology, Vanderbilt University Medical Center, Nashville, TN
37232-0443, USA.
*Author for correspondence ([email protected])
Accepted 14 May 2010
producing  cells either from dedifferentiation of mature cells or
from an unknown precursor. However, lineage tracing
experiments that can follow the fate of different cell types have
yielded conflicting results (Desai et al., 2007; Dor et al., 2004;
Inada et al., 2008; Xu et al., 2008).
The need to provide new sources of insulin-producing  cells for
diabetic patients has sparked considerable interest in finding ways
to develop increased numbers of  cells either in vivo or in vitro.
Although considerable work has indicated that  cells can replicate
to increase  cell mass (Dor et al., 2004; Nir et al., 2007; Teta et
al., 2007), the ability of other cells to give rise to new  cells has
been controversial. Thus far, the most conclusive example of  cell
neogenesis, or  cells arising from another cell type in the adult
pancreas, comes from gene therapy experiments, in which three
genes that regulate  cell development were introduced into
pancreatic acinar cells, effectively converting many acinar cells to
insulin-producing cells (Zhou et al., 2008). The enzyme-producing
acinar cells are the most abundant cells of the pancreas, making
them an attractive source for  cell replacement. We have shown
previously that acinar cells can transdifferentiate into ductal cells
in vitro in response to transforming growth factor  (TGF) and
that this is dependent upon its receptor, the epidermal growth factor
receptor (EGFR) (Means et al., 2005). However, without directed
transcriptional reprogramming, it remains to be determined whether
acinar cells have an innate ability to transdifferentiate into insulinproducing cells. We have now addressed this issue by doing direct
lineage tracing to determine the cell of origin both for hyperplastic
pancreatic ducts and for the insulin+ cells that arise within them in
vivo in response to TGF. We have found that acinar cells are
capable of transdifferentiating into hyperplastic ductal cells, but the
insulin+ cells embedded within this ductal epithelium arise from
pre-existing insulin+ cells that become intercalated into the ductal
epithelium. Thus, even though one cell type is physically
associated with another cell type, their origins might be completely
different.
DEVELOPMENT
KEY WORDS: Beta cell neogenesis, Growth factor signaling, Transdifferentiation, Mouse
2290 RESEARCH ARTICLE
MATERIALS AND METHODS
Animals
All experiments were done with approval of the Vanderbilt Institutional
Animal Care and Use Committee. Tissue-specific Cre transgenes were bred
into mice carrying the R26R-EYFP [Gt(ROSA)26Sortm1(EYFP)Cos] reporter
(Srinivas et al., 2001) and the MT-TGF transgene (Sandgren et al., 1990)
that contains the metallothionein promoter driving expression of TGF. The
Cre transgene and the R26R-EYFP reporter allele were never derived from
the same parent as this led to embryo-wide recombination in tissues
throughout the mouse. Cre transgenes included elastase500-Cre, containing
500 bp of the elastase promoter that has been shown to direct acinar-specific
expression (Kruse et al., 1993) and with growth hormone 3⬘ untranslated
sequences (Postic et al., 1999); Villin-Cre (el Marjou et al., 2004), a kind gift
from Sylvie Robine; Pdx1PB-CreERT (Zhang et al., 2005), containing the 1
kilobase islet-specific enhancer region from the Pdx1 promoter and the hsp68
minimal promoter driving expression of Cre fused to a mutated estrogen
receptor hormone-binding domain that renders Cre inactive until tamoxifen
administration; and RIP-CreERT (Dor et al., 2004) containing the rat insulin
promoter driving expression of a similar tamoxifen-inducible CreERT
protein. For these mice, tamoxifen or corn oil vehicle alone was administered
at 4-6 weeks of age, in 3 doses over a 5-day time period, 2 mg/dose via
intraperitoneal injection. One week following the final injections, mice were
placed on water containing 25 mM ZnSO4 to induce expression of the MTTGF promoter. No ductal hyperplasia was observed until 2-3 months of
Zn2+ treatment. Following 6 months of ZnSO4 treatment, mice were
euthanized and analyzed for EYFP expression.
Immunofluorescence
Pancreatic samples were fixed overnight at 4°C in 4% paraformaldehyde.
Shorter fixation times resulted in loss of EYFP, particularly in cytoplasm.
Tissues were then washed, dehydrated through a series of ethanol and
Histoclear (National Diagnostics, Atlanta, GA, USA) washes and then
embedded in paraffin. Five micron sections were collected, dewaxed and
heated either at 60°C overnight or at high pressure in a pressure cooker
(Cuisinart, East Windsor, NJ, USA) for 15 minutes in 100 mM Tris, pH 10,
cooled, washed in PBS, blocked in 5% donkey serum in PBS and then
incubated with indicated antibodies to GFP (Invitrogen, Carlsbad, CA or
Novus Biological, Littleton, CO, USA), insulin (Linco Research, St
Charles, MO, USA), glucagon (Linco Research), pancreatic polypeptide
(Linco Research), somatostatin (Santa Cruz Biotechnology, Santa Cruz,
CA, USA), amylase (Santa Cruz Biotechnology), cytokeratin 19
[Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA],
Ngn3 (DSHB) and laminin (Biogenex, San Ramon, CA, USA). Antibody
staining was visualized with Cy2, Cy3 or Cy5 fluorophor-conjugated antirabbit, anti-guinea pig, anti-mouse or anti-rat antibodies (Jackson
Immunoresearch Laboratories, West Grove, PA, USA) or biotinylated antirabbit (Vector Laboratories, Burlingame, CA, USA) followed by Cy3conjugated avidin (Jackson Immunoresearch Laboratories). Nuclei were
stained with Toto3 (Invitrogen). Images were captured on an LSM510
confocal microscope (Carl Zeiss Microimaging, Thornwood, NY, USA) at
1 m optic depth. Some sections were also labeled colorimetrically using
the above primary antibodies and the Vectastain Elite ABC Kit (Vector
Laboratories) and 3,3⬘-diaminobenzidine (Invitrogen) as the peroxidase
substrate.
Development 137 (14)
Weir et al., 1993; Chen et al., 1988; Esposito et al., 2007; Phillips
et al., 2007). In order to determine if these insulin+ cells are an
integral part of the ductal epithelium, we first examined sections
from human patients who had either chronic pancreatitis or
pancreatic cancer. Whereas many insulin+ cells were located
immediately adjacent to hyperplastic ducts (data not shown), we
also found individual insulin+ cells that were continuous with the
ductal epithelium (Fig. 1A,B) and thus did not reflect independent
endocrine structures, but rather endocrine cells that were located
within ductal epithelium. We also found that mouse models
recapitulated this phenomenon. In early adenomatous lesions
induced by pancreatic expression of the KrasG12D oncogene,
insulin+ cells were also found within ductal epithelium (Fig. 1C).
Similarly, in mice overexpressing the growth factor TGF, a
growth factor often overexpressed in human pancreatic cancer
(Korc et al., 1992), insulin+ cells were found within hyperplastic
ductal epithelium (Fig. 1D).
In both human and mouse models, many more insulin+ cells
were present immediately adjacent to ducts rather than being
continuous with the cells of the ductal epithelium. In order to
distinguish whether these closely associated cells were merely
separate, juxtaposed structures or whether the insulin+ cells might
have delaminated out of ductal epithelium, we determined whether
the single insulin+ cells as well as clusters of insulin+ cells
associated with hyperplastic ducts were each part of the ductal
epithelium. We double-labeled tissue sections from TGFoverexpressing mice for insulin and laminin. Laminin is a
basement membrane protein that delineates the outer edges of
islets, normal pancreatic ducts and some hyperplastic ducts,
effectively marking the boundaries around these structures. As
expected, when insulin+ cells were continuous with duct cells
within ductal epithelium, these insulin+ cells were contained within
the same basement membrane as the surrounding ductal cells (Fig.
2A,B). Clusters of insulin+ cells that appeared to be separate but
closely juxtaposed to hyperplastic ducts were also contained within
the basement membrane of the ducts (Fig. 2C,D). Notably, there
was no laminin present separating ducts from insulin+ cells,
Lineage tracing experiments were quantified from 2 Villin-Cre, 3
elastase500-Cre, 2 RIP-CreERT and 3 Pdx1PB-CreERT mice. Results from
Villin-Cre were combined with elastase500-Cre as these lines gave
comparable results. For each mouse, 500-2000 insulin+ cells within islets
and 200-800 insulin+ cells in hyperplastic ducts were counted. Averages
and s.e.m. were determined and compared by two-tailed t-tests.
RESULTS
Insulin+ cells arise within ductal epithelium
A number of studies have reported insulin-expressing (insulin+) cell
clusters associated with pancreatic ducts, particularly with the
hyperplastic ducts that arise following damage or disease (Bonner-
Fig. 1. Insulin-expressing cells arise within hyperplastic ductal
epithelium in humans and mice. (A,B)Pancreatic sections were
immunolabeled for insulin (brown) and nuclei were counterstained
(blue) in samples from human chronic pancreatitis (A) and human lowgrade pancreatic intraepithelial neoplasia (PanIN) lesions associated
with pancreatic ductal adenocarcinoma (B). (C)Mouse model of PanIN
in which activated KrasG12D is expressed in pancreas. (D)Mouse
overexpressing the growth factor TGF. Scale bar: 50m.
DEVELOPMENT
Statistical methods
Fig. 2. Insulin+ cell clusters are contained within ductal basement
membrane. Sections from TGF-overexpressing pancreas were
immunolabeled for insulin (green) and laminin (red) as a marker of the
basement membrane, and counterstained to show nuclei (blue).
(A)Several insulin+ cells (arrow) that are continuous with the ductal
epithelium are contained within a laminin-positive basement
membrane. (B)Laminin staining alone from A. (C)A larger cluster of
insulin+ cells (arrows) is contained within a ductal basement membrane.
(D)Laminin alone from C. Scale bar: 50m.
suggesting that the insulin+ cells were an integral part of the ductal
epithelium, possibly in the process of delaminating out of that
epithelium as happens during embryonic development.
For comparison, we also examined the formation of basement
membrane as endocrine cells normally delaminated from ductal
epithelium during late embryonic development. At embryonic day
17.5, laminin marked basement membrane around forming ducts
and islets (see Fig. S1 in the supplementary material). However, at
discreet points, no laminin was detected between the ducts and
forming islets. At these points, laminin formed a continuous layer
from the duct, extending out around the forming islet. This
continuity of basement membrane from duct to delaminating
endocrine cells is similar between embryonic tissue and TGFinduced hyperplastic tissue. However, in the TGF-overexpressing
mice, areas of contact between duct and islet clusters that were
devoid of laminin were much broader than in embryonic pancreas.
For example, approximately 12 endocrine cells were juxtaposed to
ductal epithelium in Fig. 2C, whereas only 1-3 endocrine cells were
immediately juxtaposed to duct cells in embryonic pancreas (see
Fig. S1 in the supplementary material). As larger ductal endocrine
clusters were observed in TGF-overexpressing mice, they tended
to wrap around the duct, maintaining a close association rather than
separating from the ductal epithelium. This morphology suggests
that if endocrine cells are indeed delaminating from ductal
epithelium, they remain within the original basement membrane
structure for some period of time.
Acinar cells transdifferentiate into duct cells but
not endocrine cells
Although the juxtaposition of single insulin+ cells and insulin+ cell
clusters to ductal epithelium suggested that these endocrine cells
might have arisen from the same progenitors as the hyperplastic
duct cells, we used genetic lineage tracing to define the precise cell
of origin for both the hyperplastic ducts and the insulin+ cells
associated with them. In order to label large numbers of specific
cell types in vivo, we made use of the R26R-EYFP allele, which is
RESEARCH ARTICLE 2291
transcriptionally silent until Cre recombinase removes a
transcriptional stop cassette allowing expression of the EYFP
protein (Srinivas et al., 2001). Once recombined, EYFP is
expressed from the ubiquitously active Rosa26 locus, even if Cre
is subsequently lost from the cell. Because expression of EYFP
results from genomic recombination of the R26R-EYFP allele, it
represents a heritable genetic trait that will be durably expressed
throughout the life of the cell and also passed on to any progeny
cells. Thus, tissue-specific activation of the R26R-EYFP allele
provides an indelible marker of both Cre-expressing cells and of
any cells that arise from those cells. We employed two Cre
transgenes, Villin-Cre (el Marjou et al., 2004) and elastase500-Cre
to determine the fate of acinar cells in response to TGF. We
previously showed that Villin-Cre activity is specific to acinar cells
in the pancreas, although it is also expressed in other cells of other
organs (Means et al., 2005). We also used 500 basepairs of the
elastase promoter (Kruse et al., 1993) to drive acinar expression of
Cre in the elastase500-Cre transgene. However, as mice aged, we
observed a small degree of Cre activity in endocrine cells, with
approx. 5% of endocrine cells being labeled in addition to virtually
all acinar cells in both Villin-Cre and elastase500-Cre mice.
Endocrine recombination was lower in young mice (Means et al.,
2005) (see Fig. S2 in the supplementary material) and probably
resulted from rare misexpression of the transgenes during the 8month timecourse of our experiments. However, we cannot rule out
the possibility that there is a low rate of transdifferentiation of
acinar cells into islet cells in normal pancreas.
The Villin-Cre or elastase500-Cre transgenes along with the
R26R-EYFP reporter were bred into mice expressing the MTTGF transgene. Mice were placed on water containing 25 mM
ZnSO4 to induce expression from this metallothionein promoter
and were examined 6 months later to determine if acinar cells had
given rise to the hyperplastic ductal cells and/or duct-associated
insulin+ cells. Confirming previous work showing that acinar cells
can transdifferentiate into ducts in vitro (Means et al., 2005),
hyperplastic ductal cells carried the EYFP lineage label, indicating
that acinar cells gave rise to the majority of hyperplastic ducts that
developed in response to TGF expression (Fig. 3). However, with
both acinar cell-specific Cre transgenes, there was no EYFP label
present in the majority of insulin-producing cells found either
within the hyperplastic ducts or in normal islet structures (Fig. 4).
As discussed above and quantified below, both elastase500-Cre and
Villin-Cre labeled 5.4±1.9% of normal islet  cells and a similar
5.3±1.1% of duct-associated insulin+ cells. When single insulin+
cells were observed in hyperplastic ducts, they were continuous
with EYFP+ ductal cells. Thus, acinar cells were not the cells of
origin for ductal insulin+ cells even though they gave rise to duct
cells immediately adjacent to these insulin+ cells. These data
indicate that although acinar cells can transdifferentiate into ductal
cells, they do not seem to be capable of giving rise to insulinproducing cells in response to TGF.
Duct-associated insulin+ cells arise from
pre-existing endocrine cells
Although it was possible that either duct cells or an unknown
progenitor cell might have given rise to duct-associated insulin+
cells, we first sought to rule out the possibility that pre-existing islet
cells could have intercalated into the epithelium of the hyperplastic
ducts. The Pdx1PB-CreERT transgene is expressed specifically in
islets, principally in  cells but also in 4-5% of glucagon-producing
 cells and somatostatin-producing  cells (Zhang et al., 2005).
However, Pdx1PB-CreERT is only active following treatment with
DEVELOPMENT
Lineage tracing ductal insulin+ cells
2292 RESEARCH ARTICLE
Development 137 (14)
Fig. 3. Acinar cells contribute to hyperplastic ducts. In TGF-overexpressing mice, acini were labeled for expression of the EYFP protein via the
Villin-Cre transgene and R26R-EYFP reporter. Mice were placed on Zn2+-containing water at 6 weeks of age and hyperplastic ducts were allowed to
develop for 6 months. (A)Co-immunostaining demonstrated that EYFP (green) was expressed in amylase-positive acinar cells (red) as expected, but
not all EYFP-positive cells co-expressed amylase, indicating that some previously labeled acinar cells were no longer acinar cells. (B)Amylase staining
alone from A. (C)EYFP staining alone from A. (D)EYFP was also expressed in cytokeratin-19-postive (red) hyperplastic ducts, but not in cytokeratin19-positive ducts of normal morphology (arrowheads), indicating that hyperplastic ducts, but not normal ducts, arose from acinar-to-ductal
transdifferentiation. (E)Cytokeratin 19 staining alone from D. (F)EYFP staining alone from D. Arrows, co-labeled cells; arrowheads, normal
pancreatic duct; asterisks, non-specific staining of luminal duct contents. Scale bar: 50m.
cells that were present at the time of tamoxifen injection. The
number of ductal insulin+ cells expressing EYFP reflected the
percentage labeled in islets. That is, 64.2±1.2% of islet  cells
were labeled, whereas 63.1±3.4% of duct-associated insulin+
cells were labeled. Supporting a  cell origin for ductal insulin+
cells, we did not detect any Ngn3 expression in MT-TGF mice
nor did ductal insulin+ cells co-express ductal markers such as
cytokeratin 19 (Fig. 5) or bind DBA (data not shown). We also
determined whether  cells could act as multipotent progenitor
cells and give rise to other cell types using Pdx1PB-CreERT to
label  cells. No EYFP-labeling was found in duct cells, acinar
cells or the majority of glucagon-expressing (glucagon+) cells,
both in islets and in hyperplastic ducts (Fig. 5; data not shown).
A very small number of glucagon+ cells containing the EYFP
lineage label were seen in both islets and in ductal structures,
Fig. 4. Acinar cells do not give rise to duct-associated insulin+
cells. Acinar cells were labeled using the Villin-Cre transgene to activate
EYFP expression. Following induction of hyperplastic ducts, sections
were co-immunostained for EYFP (green) and insulin (red) and nuclei
were counterstained in blue. (A,C)Although ductal cells immediately
surrounding insulin+ cells were lineage-labeled with EYFP expression,
ductal insulin+ cells (arrows) were not labeled. (B,D)EYFP only staining
of A and C, respectively. Scale bar: 50m.
DEVELOPMENT
tamoxifen, thus any endocrine cells that arise after tamoxifen has
been cleared from the bloodstream will not be recombined or
labeled with EYFP. Thus, we could investigate whether insulinproducing cells within ducts were derived from islet cells that were
present before the onset of ductal hyperplasia. We also used a
second islet-specific Cre transgene, RIP-CreERT (Dor et al., 2004)
but found that, over the 8-month timecourse of these experiments,
this transgene mediated recombination in more than half of all
insulin+ cells, even in the absence of tamoxifen (data not shown).
Additionally, RIP-CreERT labeled a relatively large percentage of
acinar cells. We examined two RIP-CreERT; R26R-EYFP mice,
not overexpressing TGF, at 7-8 months of age (the average age
of MT-TGF mice examined), and found that 3-5% of all acinar
cells were labeled with EYFP (see Fig. S3 in the supplementary
material). Therefore, we did not use the RIP-CreERT transgene for
further lineage tracing experiments. Pdx1PB-CreERT, conversely,
did not show any labeling of acinar cells in 7- to 8-month-old mice
(see Fig. S3 in the supplementary material) and labeled only
0.31±0.09% of endocrine cells with no tamoxifen over that same
timecourse (n3 mice, 2000-5000 cells counted for each).
We interbred mice carrying the islet-specific Pdx1PB-CreERT
transgene to mice carrying the R26R-EYFP reporter and the MTTGF transgene. These mice were injected with three low doses
of tamoxifen at 1 month of age to yield EYFP labeling in
approximately 60-65% of islet  cells. At this stage, MT-TGF
mice have a morphologically normal pancreas, and require at
least 2-3 months of zinc induction before hyperplastic ducts
appear. This slow development precluded the inadvertent
labeling of insulin+ cells as they arose in hyperplastic ducts, so
that only pre-existing insulin-producing  cells would be labeled.
As reported previously (Zhang et al., 2005), no labeling was seen
in non-endocrine cells. One week after the final tamoxifen
injection, mice were placed on water containing zinc to induce
TGF expression. After 6 months of zinc induction, mice were
analyzed for EYFP expression in different pancreatic cell types
(Fig. 5). As discussed above, EYFP labeling was found only in
islets in control mice not carrying the TGF transgene. However,
in TGF-overexpressing mice, insulin+ cells found either within
or closely associated with the ductal epithelium also carried the
EYFP label, indicating that these cells arose from the endocrine
Lineage tracing ductal insulin+ cells
RESEARCH ARTICLE 2293
Fig. 5. Ductal insulin+ cells arise from pre-existing  cells.
 cells were labeled via the Pdx1PB-CreERT transgene with
activity induced by tamoxifen injection. One week following
the final tamoxifen injections, mice were given Zn2+ to induce
TGF expression and analyzed 6 months later for expression
of EYFP in different cell types. (A-D)EYFP (green) was found in
single insulin+ cells (red; arrow) continuous with ductal
epithelium (cytokeratin 19, blue), indicating that the insulin+
cell arose from a pre-existing  cell. Note that a second
insulin+ cell was not labeled by EYFP, reflecting the ~65%
labeling efficiency for the Pdx1PB-CreERT transgene. A is the
merge of panels B-D. (E-H)A cluster of insulin+ cells that is in
contact with the ductal lumen was also labeled with EYFP,
indicating that those cells arose from pre-existing  cells. E is
the merge of panels F-G. Arrows indicate locations that can be
compared across rows of panels. Scale bar: 10m.
population present in these large ducts of the pancreas. Therefore,
we examined the occurrence and distribution of duct-associated
insulin+ cells in MT-TGF mice. We found that every cross-section
of the pancreas, not just in main duct regions, contained insulin+
cells within hyperplastic ducts, with an average of 12±1.3 ducts per
cross-section containing insulin+ cells (n5 MT-TGF mice, 2-3
sections counted per mouse). Thus, it is unlikely that TGFoverexpression expanded the number of insulin+ cells normally
present in ductal epithelium.
Fig. 6. Pre-existing  cells, not acinar cells, give rise to ductal
insulin+ cells in response to TGF signaling. Quantification of the
results from Figs 4 and 5 compare the ability of acinar cells and islet
cells to give rise to duct-associated insulin+ cells. For acinar lineage
tracing (left two bars), results from 3 elastase500-Cre and 2 Villin-Cre
mice were averaged. Note that although these transgenes labeled
virtually all acinar cells, they also labeled ~5% of islet (gray bar)  cells
by the time mice were 8 months old. Similarly, 5% of duct-associated
(black bar) insulin+ cells were labeled in these mice. For islet lineage
tracing (right two bars), 3 Pdx1PB-CreERT mice were used to label islet
cells with tamoxifen injected one week before MT-TGF transgene
induction and ~2 months before the appearance of hyperplastic ducts.
At the tamoxifen dosage used, 64.2±1.2% of islet (gray bar)  cells
were labeled with the EYFP lineage reporter. Similarly, 63.1±3.4% of
duct-associated (black bar) insulin+ cells were labeled. Together, these
data indicate that acinar cells do not contribute to ductal insulin+ cells
and that the majority of duct-associated insulin+ cells arise from preexisting  cells. Percentages were determined from 3 elastase500-Cre, 2
Villin-Cre and 3 Pdx1PB-CreERT mice, with 500-2000 islet insulin+ cells
and 200-800 ductal insulin+ cells counted per mouse.
DEVELOPMENT
reflecting the small number of glucagon+ cells normally labeled
by the Pdx1PB-CreERT transgene. Thus, pre-existing  cells gave
rise to ductal insulin+ cells but did not give rise to other ductassociated cell types.
When the EYFP-insulin double labeling efficiency is compared
between mice labeled with acinar-specific Cres and mice labeled
with an islet-specific Cre, it is clear that pre-existing endocrine
cells, not acinar cells, gave rise to duct-associated insulin+ cells
(Fig. 6). Examining acinar-specific Cre activity alone with its lowefficiency labeling of endocrine cells leaves open the possibility
that a small percentage of endocrine cells, in islets as well as in
ducts, arose via acinar cell transdifferentiation. However, the nearly
identical labeling of endocrine cells in islets and in ducts by
Pdx1PB-CreERT without any acinar labeling indicates that any
acinar cell transdifferentation is a minor component at best. By
combining analyses of different lineages, we conclude that acinar
cells transdifferentiated into hyperplastic ductal epithelium and that
pre-existing endocrine cells became intercalated into these
structures. However, we could not rule out that insulin+ cells were
present normally in exocrine structures and maintained their
exocrine association as ductal hyperplasia occurred. Insulin+ cells
have been reported in normal ducts of rats, with a frequency that
decreases with age (Madden and Sarras, 1989). Therefore, we
examined whether insulin+ cells are present in normal pancreatic
ducts as mice age. Examining random cross-sections of pancreas
from 2-month-old normal mice, no insulin+ cells were found within
either ductal or acinar structures (n3 mice, 3-4 cross-sections
each). Because the main pancreatic duct has been proposed as a site
of  cell neogenesis (Sharma et al., 1999), we also specifically
examined sections through the main pancreatic duct. Two-monthold mice did indeed contain 1-2 clusters each of insulin+ cells
continuous with ductal epithelium per approximately 1000 ductal
cells of the main pancreatic duct or closely associated interlobular
duct branches. Each cluster comprised 1-3 insulin+ cells for an
average of 0.32±0.13% of main duct cells (see Fig. S4 in the
supplementary material; data not shown). However, in four 6month-old mice examined, we found no insulin+ cells within the
main pancreatic duct, indicating that less than 0.1% of normal main
duct cells are insulin+, which is comparable with the results from
rat pancreas (Madden and Sarras, 1989). Thus, it appears that mice
lose insulin+ cells within their larger ducts as they age. Because we
began Zn2+ induction of TGF expression at approximately 6
weeks of age, it was possible that TGF merely expanded the
DISCUSSION
We have found that pancreatic acinar cells have a limited ability to
give rise to other cell types within the adult mouse pancreas in
vivo. Using rigorous genetic lineage tracing, we have demonstrated
that pancreatic acinar cells have the ability to transdifferentiate into
the hyperplastic ducts that develop upon overexpression of TGF.
Although numerous insulin-expressing cells were part of this
hyperplastic ductal epithelium, we have shown that these ductal
insulin+ cells originated from pre-existing  cells and were not the
result of  cell neogenesis. Although acinar cells are capable of
giving rise to ductal cells in response to TGF signaling both in
vitro and in vivo, they were not the cell of origin for endocrine cells
found associated with such ducts in vivo.
Phylogenetic differences in  cell location
Although the majority of  cells are clustered in pancreatic islets
in mammals, other species show a variety of localizations of
insulin-producing cells (for a review, see Gannon and Wright,
1999). Tunicates and cephalochordates such as amphioxus have
individual insulin+ cells located within gut epithelium, providing
an evolutionary precedent for insulin+ cells within epithelial sheets.
However, vertebrates as distant as agnathans organize  cells into
distinct islet clusters. Thus, the ability of  cells to emigrate out of
epithelial sheets wherein they arise and cluster evolved even before
development of a distinct exocrine pancreas.
Acinar cells transdifferentiate into ductal cells
In addition to demonstrating that ductal insulin+ cells do not arise
from acinar cells, this study demonstrates that  cells do not give
rise to hyperplastic ducts in response to TGF-overexpression.
Rather, TGF induces transdifferentiation of acinar cells into
hyperplastic ducts. In contrast to our studies, Strobel et al. (Strobel
et al., 2007) found that acinar cells gave rise to only a small
percentage of the hyperplastic ducts that arose in response to
cerulein, a cholecystokinin analog that induces pancreatitis in mice.
This difference might result from specific effects of TGF on
acinar cells and/or because cerulein treatment results in widespread
apoptosis of acinar cells. It is probable that both acinar cells and
duct cells can give rise to hyperplastic ducts in the pancreas, with
acinar cells predominating in our study owing to their
overwhelming abundance.
Gidekel Friedlander et al. (Gidekel Friedlander et al., 2009)
performed lineage studies to demonstrate that activation of the
KrasG12D oncogene via the insulin promoter could give rise to
ductal adenomas, suggesting that islets could be a cell of origin for
pancreatic ductal adenocarcinoma. However, this group used the
RIP-CreERT transgene to activate KrasG12D expression. The
authors described little or no labeling of acinar cells with this
CreERT transgene. We have shown here that RIP-CreERT
recombines 3-6 percent of acinar cells. The discrepancy in our
studies might arise from the reporter gene used to assess
recombination. Gidekel Friedlander et al. (Gidekel Friedlander et
al., 2009) used the R26R reporter that expresses -galactosidase
(gal) upon Cre-mediated recombination (Soriano, 1999) rather
than the EYFP reporter that we used (Srinivas et al., 2001). We
have found that it is very difficult to preserve gal activity in adult
acinar cells. In our hands, reliable gal detection in acinar cells
requires transcardial perfusion of the fixative, rapid freezing and
re-fixation of tissue sections on the slide. Even EYFP is less wellpreserved in acinar cytoplasm than in islet cytoplasm (see Fig. S3
in the supplementary material) but its small size allows EYFP to
diffuse into the nucleus where it is more readily detected. Thus, the
Development 137 (14)
ability of KrasG12D to induce islet cells to give rise to ductal
adenomas will have to await experiments with a more specific islet
CreERT, such as the Pdx1PB-CreERT used here.
Origin of  cells in the adult pancreas
Various cell types have been postulated as possible sources for 
cell renewal in vivo. Many researchers have suggested that there
are facultative progenitor cells residing within normal ductal
epithelium that lead to islet cell regeneration. These studies have
primarily relied on morphological analysis after the stimulation
of pancreas-wide damage by either partial pancreatectomy
(Ackermann Misfeldt et al., 2008; Bonner-Weir et al., 1993) or
ductal ligation (Wang et al., 1995) that results in the presence of
insulin+ cells closely associated with ducts. The appearance of
these insulin+ cells has been interpreted as new islets budding from
the ductal epithelium (Esposito et al., 2007; Gu and Sarvetnick,
1993; Phillips et al., 2007; Song et al., 1999). However, a recent
report from Desai et al. (Desai et al., 2007) demonstrated that
insulin+ cells in islets observed following surgical removal of part
of the pancreas did not result from acinar cell transdifferentiation,
a result similar to ours using growth factor induction of ductal
hyperplasia. Desai et al. (Desai et al., 2007) did not determine what
the cell of origin was in their partial pancreatectomy model, nor did
they examine duct-associated insulin+ cells. In order to determine
if duct cells could transdifferentiate into insulin-producing cells,
Solar et al. (Solar et al., 2009) used the HNF1 promoter to drive
CreERT in pancreatic ducts. They found that duct cells in adult
mice did not contribute to insulin-producing cells even after
pancreatic duct ligation, but they appeared to examine only
endocrine cells in islet structures, not within ducts, and they did not
identify what the origin was for these endocrine cells. Inada et al.
(Inada et al., 2008) used the carbonic anhydrase II (CAII) promoter
to drive CreERT expression in pancreatic ducts. After partial
pancreatectomy, both insulin+ and ductal cells were labeled with
the Cre reporter, suggesting that ducts could give rise to new 
cells. However, this CAII-CreERT transgene was not characterized
for its expression pattern or labeling efficiency, thus putting any
conclusions on hold until those are determined.
Other studies have also shown that duct-associated insulin+ cells
can arise in mouse pancreas, for example, in response to
adenoviral-mediated overexpression of two EGFR ligands, HBEGF and betacellulin (Kozawa et al., 2005; Tokui et al., 2006). The
ability of both these ligands to activate EGFR, as does TGF used
in the studies presented here, suggests that the increase in  cells
might have resulted from relocalization and/or expansion of preexisting  cells.
In addition to insulin+ cells appearing in ducts, neurogenin 3
(Ngn3) has also been detected in pancreatic ducts in response to
damage. Partial pancreatectomy that retains the main pancreatic
duct in the tail of the pancreas while removing most other exocrine
and endocrine tissue resulted in the appearance of Ngn3+ cells in
ducts (Ackermann Misfeldt et al., 2008), whereas partial
pancreatectomy completely removing the distal main duct did not
result in Ngn3 expression (Lee et al., 2006). Xu et al. (Xu et al.,
2008) demonstrated that partial pancreatic duct ligation, a method
of constricting flow through the main pancreatic duct resulting in
damage distal to the site of ligation, also resulted in the appearance
of Ngn3-expressing cells. Ngn3 is a regulator of  cell neogenesis
during embryonic development, so its expression in adult pancreas
might suggest that  cell neogenesis could be occurring. In either
of the above studies it is not known which cells gave rise to the
Ngn3+ cells. No Ngn3+ cells were detected in MT-TGF mice.
DEVELOPMENT
2294 RESEARCH ARTICLE
However, cellular changes in response to TGF occur slowly over
many months so transient Ngn3 expression could have been
missed. We chose this model because we could be sure that
tamoxifen would be cleared from the bloodstream long before
ductal insulin+ cells were observed. However, we have also
examined mice that overexpress a particular isoform of a related
family member, soluble HB-EGF (sHB-EGF) (Ray et al., 2009). In
these mice, ductal hyperplasia occurs beginning around 1-2 months
of age and many ductal insulin+ cells are observed by 2 months of
age (data not shown). Even in these mice, no Ngn3 was detected.
The discrepancies in Ngn3 detection throughout the literature might
be owing to different inductive cues and/or differences in
antibodies used for detection. In none of the studies were adult
Ngn3+ cells lineage mapped to determine their cell of origin. Wang
et al. (Wang et al., 2009) have demonstrated a low level of Ngn3
expression in adult islets and have demonstrated that its loss in
adult  cells affected islet function. Therefore, Ngn3 is not only a
marker of endocrine precursors but might also be a marker of some
adult  cells.
In the studies by Xu et al., adult Ngn3-expressing cells were not
shown to give rise to  cells in vivo, but could be induced to
differentiate into insulin-producing cells ex vivo, suggesting that
adult pancreatic cells are capable of generating new  cells but the
adult environment prohibits completion of this pathway. In support
of this hypothesis, others have reported ex vivo expansion of
insulin+ cells from pancreatic cultures consisting primarily of
acinar cells (Baeyens et al., 2005; Minami et al., 2005). Thus, it
might be possible to derive ex vivo methods for increasing  cell
mass from adult pancreas for subsequent islet transplantation into
diabetic patients.
We have shown previously that acinar cells (Means et al., 2005)
isolated from mature mouse pancreas could transdifferentiate into
ductal cells ex vivo under the influence of exogenous TGF and
that this process involved a dedifferentiation step. Here we show
that this is recapitulated in vivo with acinar cells giving rise to
hyperplastic ducts. The use of transgenes, allowing for both acinar
cell- and islet cell-specific Cre recombinase expression coupled
with the R26R-EYFP reporter, enabled us to determine the level of
plasticity of adult pancreatic acinar cells in response to TGF in
vivo. Our findings that pre-existing  cells gave rise to ductassociated insulin+ cells are in agreement with the recent results of
others, that only  cells can give rise to new  cells in vivo. Dor et
al. (Dor et al., 2004) used an elegant insulin-promoter-based
approach to show that existing  cells are the sole source of new 
cells in the adult mouse under normal conditions. Along the same
lines, an innovative DNA analog-based lineage tracing method has
been utilized to demonstrate that  cell generation is the result of
self duplication during both normal growth conditions and in cases
of acute  cell expansion, and does not involve specialized
progenitors (Teta et al., 2007).
Interestingly, multiple studies that demonstrated an increase in
insulin-producing cells both in vivo and in vitro used EGFR ligands
to stimulate this  cell expansion (Baeyens et al., 2005; Minami et
al., 2005; Rooman et al., 2000; Suarez-Pinzon et al., 2005a; SuarezPinzon et al., 2005b). Although our findings indicate that EGFR
signaling does not lead to islet neogenesis in vivo, EGFR signaling
may allow alterations in the  cell environment, such as ductal
localization, that allow increased proliferation or survival. It will be
interesting to determine in future studies whether the ductal
environment promotes the proliferation of functional  cells. Such
studies could aid our ability to increase endocrine function in diabetic
patients that retained some  cell mass.
RESEARCH ARTICLE 2295
Acknowledgements
We thank Steven Leach for collaborating to generate the elastase500-Cre
mouse and for critically reading this manuscript. This work was supported by
JDRF research grant 1-2006-759 (A.L.M.), and through the Vanderbilt Digestive
Diseases Research Center (NIH P30DK058404, M.K.W.) and the use of the
Vanderbilt Cell Imaging Shared Resource (NIH CA68485, DK20593, DK58404,
HD15052, DK59637 and EY08126). Deposited in PMC for release after 12
months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.048421/-/DC1
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