1 Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN
37232, USA
2 Department of Medicine, Vanderbilt University School of Medicine, Nashville,
TN 37232, USA
3 Department of Surgery, Johns Hopkins University School of Medicine, Baltimore,
MD 21287, USA
4 Division of Gastroenterology, Department of Genetics and Abramson Cancer
Center University of Pennsylvania School of Medicine, Philadelphia, PA 19104,
USA
5 Department of Cell and Developmental Biology, Vanderbilt University School of
Medicine, Nashville, TN 37232, USA
6 Division of Endocrinology, Diabetes and Metabolism, University of Pennsylvania
School of Medicine, Philadelphia, PA 19104, USA
7 Department of Oncology, Johns Hopkins University School of Medicine,
Baltimore, MD 21287, USA
8 Department of Cell Biology, Johns Hopkins University School of Medicine,
Baltimore, MD 21287, USA
* Author for correspondence (e-mail: stleach{at}jhmi.edu)
Accepted 1 June 2005
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SUMMARY |
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Key words: Pancreas, Metaplasia, Differentiation, Transdifferentiation, Stem cells, TGF, Cancer, Mouse
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Introduction |
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The exocrine pancreas undergoes metaplastic change in the setting of both
chronic pancreatitis and pancreatic cancer. In these conditions, the pancreas
changes from an acinar cell-predominant tissue to a tissue comprised
predominantly of ductal epithelium. These metaplastic ducts have been
postulated to arise either by outgrowth of normal ductal epithelium, by
activation of pancreatic stem cells or by transdifferentiation of mature cell
types (De Lisle and Logsdon,
1990; Githens et al.,
1994
; Rooman et al.,
2000
; Sphyris et al.,
2005
). Without direct lineage tracing in combination with
molecular marker analysis, the cell of origin for metaplastic ductal
epithelium has remained controversial. Elucidating this mechanism would
represent a significant advance not only in understanding the plasticity of
terminally differentiated tissues, but also in determining the cellular basis
of pancreatic cancer, as metaplastic ducts have frequently been proposed to be
the progenitors for pancreatic ductal adenocarcinoma
(Lowenfels et al., 2000
;
Parsa et al., 1985
;
Song et al., 1999
;
Wagner et al., 2001
;
Wagner et al., 1998
).
The metaplastic conversion of acinar cells to ductal cells can be
recapitulated by culturing pancreatic epithelium in vitro. When exocrine
epithelial explants are cultured in or on an appropriate matrix, loss of
acinar cells is frequently associated with a reciprocal increase in ductal
epithelium (De Lisle and Logsdon,
1990; Githens et al.,
1994
; Rooman et al.,
2000
; Sphyris et al.,
2005
). Although a variety of culture conditions have been shown to
promote this acinar-to-ductal conversion, the molecular and cellular
mechanisms are not known. We have used this in vitro metaplastic conversion to
understand three basic processes underlying epithelial metaplasia in mammalian
pancreas: (1) to identify autocrine and/or paracrine pathways regulating
pancreatic metaplasia; (2) to identify the cell of origin for metaplastic
ductal epithelium; and (3) to identify intermediary cell populations arising
during this process. Using primary explant cultures and rigorous lineage
tracing techniques, we demonstrate that acinar cells undergo conversion to
metaplastic ductal epithelial cells in response to TGF
and EGFR
signaling; that this represents a true transdifferentiation event involving
conversion of terminally differentiated acinar cells to a ductal epithelial
phenotype; and that this transdifferentiation occurs via intermediates that
are nestin positive and simultaneously express both acinar and ductal markers.
These results provide a novel example of rigorously documented
transdifferentiation within a mature mammalian epithelium, and suggest that
plasticity of fully differentiated epithelial cells may contribute to the
generation of neoplastic precursors.
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Materials and methods |
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Preparation of epithelial explant cultures
Explant cultures of adult mouse pancreas were established by modification
of previously published protocols (De Lisle
and Logsdon, 1990; Githens et
al., 1994
; Wagner et al.,
2002
). Whole pancreas was harvested and digested in 0.2 mg/ml
collagenase-P (Boehringer Mannheim, Mannheim, Germany) at 37°C. Following
multiple washes with Hanks balanced salt solution (HBSS) supplemented with 5%
fetal bovine serum (FBS), collagenase-digested pancreatic tissue was
sequentially filtered through 500 µm and 105 µm polypropylene mesh
(Spectrum Laboratories, Laguna, CA). The filtrate was passed through a 30% FBS
cushion at 1000 rpm. The cellular pellet was resuspended in Waymouths MB 752/1
media or RPMI1640 media (Gibco BRL, Gaithersburg, MD) supplemented with
penicillin G (1000 U/ml), streptomycin (100 µg/ml) with 1% heat-inactivated
FBS (growth factor experiments) or 10% FBS (lineage tracing experiments). An
equal volume of neutralized rat tail collagen type I (RTC) (Collaborative
Biomedical Products, Bedford, MA) was added to the cellular suspension. The
cellular/RTC suspension was supplemented with 0.1 mg/ml soybean trypsin
inhibitor (Sigma Chemicals, St Louis, MO) and 1 µg/ml dexamethasone
(Sigma). Cellular/RTC suspension (500 µl) was pipetted into each well of a
24-well plate (well diameter=16mm) (Corning, Corning, NY) pre-coated with 200
µl of RTC. After solidification of the RTC, media supplemented with
penicillin G, streptomycin (100 µg/ml) and FBS (at above mentioned
concentrations) were added. Cultures were maintained at 37°C and 5%
CO2 in air for up to 14 days. Explants harvested from
non-transgenic mice were maintained in the presence or absence of recombinant
human TGF
or HGF (R&D Systems). Where appropriate, explants were
additionally treated with the EGF receptor inhibitors AG1478 (Sigma) or
EKI-785 (generously provided by Philip Frost at Wyeth-Ayerst). Media
supplemented with appropriate growth factors and/or inhibitors were exchanged
on day 1 and day 3. For detection of ß-gal activity, whole collagen gels
were fixed in 0.2% glutaraldehyde/1% formaldehyde and stained in X-gal
overnight at 37°C (Means et al.,
2003
).
Assessment of cell death and cell proliferation
Cell viability was determined by Trypan Blue exclusion. Immediately prior
to plating, an aliquot of cells were mixed with an equal volume of 0.4% Trypan
Blue stain (Gibco) for 10 minutes, washed and counted for the number of blue
(dead) and non-blue cells (alive). After 3 days of culture, cells suspended in
collagen were digested with 25 µg/ml collagenase P for 10 minutes, washed
and stained with Trypan Blue as above. Four different experiments were
performed, with 1000-2000 cells counted in each sample. Results are presented
as mean±s.e.m. For assessment of cell proliferation, BrdU was added to
the culture medium throughout the 5 days of culture, and explants were
similarly analyzed for BrdU incorporation using immunofluorescence.
RNA extraction and semi-quantitative RT-PCR analysis of nestin expression from total cellular RNA was performed using TRIZOL Reagent (Life Technologies, Rockville, MD). cDNA was prepared by random priming from 1 µg of total RNA using a First-Strand cDNA Synthesis kit (Life Technologies, Rockville, MD) according to the manufacturer's instructions. Amplification was carried out in 50 µl of reaction mixture containing dNTP (200 µM each), 30 pmol of each of the primers and 2.5 U of Taq DNA polymerase (Qiagen). For nestin, Gapdh and ß-actin amplifications, 5 µl of cDNA template was amplified using the following primer pairs: nestin (annealing temp: 59°C), forwards 5'-GCT GGA ACA GAG ATT GGA AGG C-3' and backwards 5'-TCA AGG GTA TTA GGC AAG GGG G-3'; GAPDH (annealing temp: 58°C) forwards 5'-TGT TCC AGT ATG ACT CCA CTC ACG-3' and backwards 5'-GCC CTT CCA CAA TGC CAA AG-3'; ß-actin (annealing temp: 59°C), forwards 5'-GCT CGT CGT CGA CAA CGG CTC-3' and backwards 5'-CAA ACA TGA TCT GGG TCA TCT TCT-3'.
PCR product accumulation was assessed at 20, 25 and 30 cycles of amplification in order to confirm linear detection of PCR product. The expected and observed amplification product sizes were as follows: nestin, 372bp; GAPDH, 384 bp; ß-actin, 359 bp.
Immunostaining
The following antibodies were used for immunofluorescence analysis: rabbit
polyclonal anti-nestin (gift from Dr R. McKay), mouse monoclonal anti-nestin
(Pharmingen), goat polyclonal anti-amylase (Santa Cruz Biotechnology), rabbit
polyclonal anti-cytokeratin, wide spectrum (Dako), rabbit polyclonal
anti-carbonic anhydrase II (Chemicon) and sheep polyclonal keratin 19 (The
Binding Site). For immunofluorescent labeling of explanted pancreatic tissue,
collagen gels containing explanted pancreas were fixed in 4:1 methanol:DMSO
overnight, 4°C, then washed and stored at 20°C in 100%
methanol. Cultures in collagen disks were rehydrated, washed in PBS, then
PBSBT (PBS+ 0.5% tritonX-100 + 2% BSA). Disks were blocked with 5% normal
donkey serum in PBSBT for 2 hours at room temperature, then incubated
sequentially with the primary and secondary antibodies diluted in PBSBT,
overnight at 4°C. Following each antibody, disks were washed extensively
in PBT (PBS + 0.5% tritonX-100). After the final overnight incubation, the
cultures were washed twice in PBT, three times in PBS, then counterstained
with YoPro nuclear dye (Molecular Probes) and washed in PBS. Images were
captured on a Zeiss LSM-510 Meta confocal microscope at an optical depth of 1
µm. Immunoperoxidase staining was performed on paraffin-embedded tissue
that was sectioned at depths of 2 or 5 µm, using the Vectastain ABC Elite
kit (Vector Labs) as indicated by manufacturer. Antibodies used were rabbit
anti-amylase (Sigma) and rabbit anti-cytokeratin, wide spectrum (Dako). For
quantitative analysis of immunohistochemically and histochemically stained
cells, all quantification is presented as mean±s.e.m. For each
analysis, cells were counted from at least three independent experiments for a
total of 900 to 1600 cells counted per analysis.
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Results |
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|
Based on the ability of hepatocyte growth factor (HGF) to regulate
epithelial differentiation and morphology in a variety of settings
(Brinkmann et al., 1995), as
well as the recent implication of the HGF receptor as an a marker of
pancreatic epithelial precursors (Suzuki
et al., 2004
), we next tested this growth factor for its ability
to induce acinar-to-ductal metaplasia. Similar to the effects of rhTGF
,
treatment of epithelial explants with human recombinant HGF also induced loss
of acinar cells, and replacement by metaplastic ductal epithelium in a
dose-dependent manner (Fig.
1H,J).
These data demonstrate that two growth factors, acting on different
tyrosine kinase receptors, each induce similar effects on pancreatic
epithelial explants. In many systems, signaling through EGFR is influenced by
other receptors that mediate the cleavage and activation of EGFR ligands
(Prenzel et al., 1999;
Uchiyama-Tanaka et al., 2002
).
Therefore, we examined the requirement of EGFR signaling in the generation of
metaplastic epithelium in response to either TGF
or HGF. For these
experiments, acinar cultures were treated with either TGF
or HGF in the
presence or absence of the EGFR/erbB2 tyrosine kinase inhibitors EKI-785
(Discafani et al., 1999
) and
AG1478 (Lin et al., 1997
). As
expected, these inhibitors caused a dose-dependent decrease in
TGF
-induced ductal metaplasia, with complete inhibition observed at a
concentration of 1.0 µM EKI-785 (Fig.
1K). By contrast, EKI-785 had a limited effect on generation of
metaplastic epithelium by HGF, with even 1.0 µM EKI-785 unable to
completely abolish the response to HGF
(Fig. 1K). At a concentration
of 5.0 µM, AG1478 showed an identical effect (data not shown), inhibiting
TGF
-induced but not HGF-induced acinar-to-ductal metaplasia. The
limited inhibition of HGF activity in response to EGFR inhibitors suggests
that HGF may act partially upstream of EGFR; more importantly, these data
indicate that separate signaling pathways, perhaps converging downstream, can
independently induce a metaplastic change in pancreatic epithelial
differentiation.
Metaplastic ductal epithelium arises by acinar cell transdifferentiation
Because acinar cells are lost during ductal metaplasia of the pancreas, it
has frequently been proposed that transdifferentiated acinar cells represent
the source of metaplastic ductal epithelium. However, tracing the fate of
acinar cells either in vivo or in vitro has been complicated by extensive cell
death, which typically occurs during the process of acinar-to-ductal
metaplasia. To trace the fate of any remaining acinar cells, large number of
cells needed to be labeled in an entirely cell type-specific manner. To
generate such a label, we developed two different methods for acinar-specific
recombination of the R26R reporter allele in
Gt(ROSA)26Sortm1Sor (R26R) mice
(Soriano, 1999). The R26R
lacZ reporter allele is silent until a transcriptional stop cassette
is excised by Cre-mediated recombination. Once recombined, the lacZ
gene is expressed from the ubiquitously active Rosa26 locus, even if Cre is
subsequently lost from the cell. As expression of ß-gal enzymatic
activity results from genomic recombination of the R26R 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, acinar-specific
activation of the R26R allele provides an indelible marker of both acinar
cells and of any cells that arise from acinar cells.
To obtain acinar-specific recombination of the R26R allele, mice expressing a Villin-Cre transgene were crossed onto the R26R reporter line. Although the Villin-Cre transgene induced recombination in multiple tissues including intestine and kidney, ß-gal activity in pancreatic tissue from Villin-Cre;R26R mice was strictly confined to acinar cells. No ß-gal activity was seen in other pancreatic cell types, including islet and ductal cells (Fig. 2A-D; see Fig. S1A,B in the supplementary material). Even the terminal intercalated ducts most closely associated with acini were negative for ß-gal activity. In addition, no ß-gal activity was seen in single transgenic mice carrying either the Villin-Cre transgene or the R26R allele alone (data not shown).
To determine whether acinar cells could transdifferentiate into ductal
cells, acinar-enriched epithelial explants were isolated from pancreas of
Villin-Cre;R26R mice and cultured in the presence of rhTGF. At the time
of isolation, ß-gal staining was observed exclusively in acinar cells,
with 98.0±0.6% of ß-gal-positive cells also staining positive for
amylase (Fig. 3A,C). No
ß-gal activity was observed in cells expressing duct-specific keratins
(Fig. 3E). Cytokeratin-positive
ductal epithelial cells represented 15.0±2.8% of the initial cell
population in freshly harvested explants, and probably represented terminal
intercalated ductal epithelium based on characteristic squamous morphology and
location in the center of acinar cell clusters. These results confirm our in
vivo observation that Villin-Cre;R26R provides a genetic lineage label
specific to acinar cells and their progeny.
|
Although Villin-Cre;R26R lineage tracing revealed that acinar cells could
transdifferentiate into ductal cells, it was possible that the initially
isolated epithelium contained small numbers of ß-gal-positive,
amylase-positive cells that were not fully differentiated and thus capable of
changing their differentiation pathway. To assure that a small number of
uncharacterized cells did not represent the source of metaplastic ductal
epithelium, we examined the amount of proliferation occurring during the
culture period. BrdU was added to the culture medium throughout the 5 days of
culture, and explants were then assayed for the number of cells that had
undergone proliferation, as determined by BrdU incorporation. During the 5
days of culture, only 10.3±3.5% of cells incorporated BrdU. Thus, it is
unlikely that expansion of ductal epithelium occurred by proliferation of a
small population of uncharacterized cells. Rather, it appears that the
majority of acinar cells are capable of undergoing transdifferentiation
without obligate intervening cell division. Similar observations have recently
been reported by Sphyris and colleagues
(Sphyris et al., 2005).
|
In order to further implicate mature acinar cells as the source of
metaplastic epithelium, we performed additional experiments using an inducible
Cre system, so that genetic labeling of acinar cells could be delayed until
adulthood. For these experiments, acinar cells were genetically labeled using
a tamoxifen-inducible Cre driven by the Elastase (Ela) promoter. Ela-CreERT2
transgenic mice express a fusion protein comprised of Cre recombinase and a
modified estrogen receptor ligand binding domain (ERT2) under control of the
acinar cell-selective elastase promoter (D.A.S., unpublished). Under normal
conditions, this fusion CreERT2 is inactive, apparently owing to cytoplasmic
sequestration by heat-shock proteins. However, upon tamoxifen binding, the
CreERT2 fusion protein translocates to the nucleus, resulting in effective Cre
activity. When Ela-CreERT2 transgenic mice were crossed with R26R mice
(Soriano, 1999) no leaky
lacZ expression was noted in the absence of tamoxifen induction
(D.A.S., unpublished). Following intraperitoneal tamoxifen injection,
pancreatic tissue from Ela-CreERT2; R26R mice demonstrated acinar
cell-selective ß-gal activity, with no activity observed in inter- or
intra-lobular ducts, islets or stroma (D.A.S., unpublished; see Fig. S1 in the
supplementary material). Activity in acinar cells was mosaic, inducing
recombination of the R26R reporter in
40% of acinar cells, suggesting
that, under the conditions employed, tamoxifen was only able to induce Cre
activity in a limited number of cells. Following a seven-day in vivo `pulse'
of tamoxifen and a subsequent seven-day `chase' period to ensure complete
tamoxifen clearance, epithelial explants were harvested and subjected to
TGF
-induced acinar-to-ductal metaplasia followed by staining for
ß-gal activity. Consistent with observations made on intact tissue,
freshly harvested epithelial explants from tamoxifen-treated mice displayed a
mosaic distribution of ß-gal activity restricted to acinar cells
(Fig. 4A,B). When epithelial
explants were isolated and cultured in the presence of TGF
, the
duct-like epithelial cells that arose were found to stain with X-gal in a
similar mosaic pattern (Fig.
4C-F), confirming the acinar cell origin of metaplastic ductal
epithelium and identifying transdifferentiation as an active mechanism driving
tissue metaplasia in this system.
|
|
The onset of nestin expression coincided with the dual expression of
acinar-specific amylase and duct-specific cytokeratins. Reflecting in vivo
expression patterns, amylase and cytokeratins were not co-expressed at the
time of initial explant isolation (Fig.
6G). However, by day 2 of culture in TGF, cell clusters
that were beginning to form expanded lumena contained many cells that
co-expressed amylase and ductal cytokeratins
(Fig. 6H). By day 5 of culture,
there were no remaining amylase-positive cells and most cells within cystic
clusters expressed ductal cytokeratins
(Fig. 6I). However, although
ductal cytokeratins began to be expressed prior to the loss of amylase
protein, another marker of ductal epithelium, carbonic anhydrase II, was not
detected until after the loss of amylase
(Fig. 6J-L). These findings
suggest that metaplastic conversion from an amylase-expressing, acinar
cell-predominant population to a cytokeratin-expressing duct cell-predominant
population occurs via nestin-positive intermediates that gradually lose
amylase expression while progressively gaining cytokeratin and then carbonic
anhydrase II expression.
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Discussion |
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This conclusion is further supported by our analysis of cell death and cell proliferation. Some 90% of emerging ductal elements failed to incorporate BrdU during the 5-day culture period, suggesting that expansion of this ductal epithelium did not require cell proliferation. Although we did see extensive cell death, common to most primary cell cultures, the most viable cell isolates had only one-third of cells dying by day 3 of culture, a time at which loss of acinar identity and acquisition of a ductal phenotype are already observed. Owing to the significant extent of cell death observed during acinar-to-ductal metaplasia, it is possible that not all acinar cells have the capacity to transdifferentiate. However, the ability of 35-67% of cells to survive and undergo ductal differentiation suggests that a rather large subset of acinar cells carry this capability.
Transdifferentiation in other tissues
During development, undifferentiated, tissue-specific progenitor cells
retain considerable plasticity, as evidenced by multiple examples of
transdetermination, in which local tissue-specific progenitors undergo
reprogramming to generate a variety of alternate cell fates. In developing
mouse pancreas, transdetermination from a pancreatic to an intestinal cell
fate has been demonstrated by following the fate of presumptive pancreatic
progenitors in the presence and absence of the bHLH transcription factor,
Ptf1a/p48. These studies have demonstrated that, in the absence of functional
Ptf1a/p48 protein, cells normally fated to become pancreas instead become
mature duodenal cells (Kawaguchi et al.,
2002). Similary, Drosophila imaginal discs and avian limb
buds also appear to be characterized by a considerable capacity for
transdetermination (Maves and Schubiger,
1999
; Takeuchi et al.,
1999
).
In contrast to these examples of plasticity involving undifferentiated cell
types, transdifferentiation represents the conversion of one fully
differentiated cell type to another (Shen
et al., 2003). To date, rigorously documented examples of
transdifferentiation occurring in the context of adult epithelium have largely
been confined to amphibian species, with lens regeneration by retinal
pigmented epithelial cells representing the most frequently cited example
(reviewed by Eguchi and Kodama,
1993
). Transdifferentiation is also apparent during appendage
regeneration in urodele amphibians, in which tissue within the wound blastema
undergoes dedifferentiation followed by multiple rounds of cell division, with
subsequent redifferentiation along a variety of lineage pathways. For example,
labeling of individual radial glial cells following tail amputation in axolotl
salamanders has demonstrated transdifferentiation of these ectodermally
derived cells to generate new muscle and cartilage
(Echeverri and Tanaka,
2002
).
Although frequently reported, instances of transdifferentiation in
mammalian systems are more difficult to evaluate. These examples frequently
involve changes in differentiation of clonal cell lines in response to a
variety of genetic and epigenetic influences. In these systems,
transdifferentiation has typically been defined by clonal dilution or
individual examination of isolated cells undergoing biochemical and
morphological conversion to a different cell type. For example, mouse
myoblasts have been reported to transdifferentiate into adipocytes
(Hu et al., 1995), murine
melanoma cells to glial cells (Slutsky et
al., 2003
) and rat acinar cell carcinoma cells to hepatocytes
(Shen et al., 2003
;
Shen et al., 2000
). However,
it remains unclear how well these single cell observations faithfully
recapitulate events occurring in an intact epithelium, and it is notable that
each of these examples appears to involve input cells with a heightened
progenitor potential. In the case of mouse myoblasts, the observed phenomenon
might better be described as transdetermination of a tissue-specific
precursor. Similarly, the observation of apparent transdifferentiation in
mouse melanoma and rat acinar cell carcinoma cells might simply reflect
heightened plasticity associated with the malignant phenotype.
Acinar cell transdifferentiation as the mechanism for acinar-to-ductal metaplasia
In the current study, we have shown that fully differentiated exocrine
cells in adult mouse pancreas are capable of undergoing transdifferentiation,
and that this transdifferentiation event represents the cellular mechanism for
induction of acinar-to-ductal metaplasia. We have used morphological and
molecular characterization as well as genetic labeling to clearly define a
direct lineage relationship between acinar cell precursors and their ductal
progeny. We have shown that this genetic lineage label is initially expressed
exclusively by fully differentiated acinar cells, as judged both by columnar
acinar morphology and by co-labeling with two molecular markers, amylase and
carboxypeptidase A (Fig. 3;
data not shown). We have also rigorously defined a ductal epithelial phenotype
within the final population of ß-gal-labeled cells, based on classical
cuboidal or simple squamous morphology and expression of both ductal
cytokeratins and carbonic anhydrase II. The direct lineage relationship
between these cell types was clearly established by tracing heritable
ß-gal activity arising from acinar cell-specific recombination of the
R26R reporter allele, accomplished by both the Villin-Cre and Elastase-CreERT2
transgenes. Thus, we can firmly conclude that mature acinar cells can
transdifferentiate to form metaplastic ductal epithelium. We further conclude,
based on an observed low frequency of cellular proliferation, that this
transdifferentiation does not require intervening cell division. Based on
previously established criteria (Eguchi and
Kodama, 1993; Shen et al.,
2003
), we conclude that the current results represent a unique
example of rigorously documented transdifferentiation occurring in mature
mammalian epithelium.
Acinar-to-ductal transdifferentiation occurs via a dedifferentiated intermediate cell type
During lens or tail regeneration in amphibia, transdifferentiation
typically proceeds by way of dedifferentiated intermediates
(Echeverri and Tanaka, 2002;
Eguchi and Kodama, 1993
). In
the current study, the detection of nestin expression in intermediary cells
suggests that acinar cell transdifferentiation may involve a similar
undifferentiated intermediate. Although the use of nestin as a label for
undifferentiated pancreatic epithelial progenitors remains controversial,
recent studies have confirmed that nestin-expressing epithelial cells are
indeed present during early pancreatic development, and that these cells
represent the cell of origin for differentiated exocrine cells
(Delacour et al., 2004
;
Esni et al., 2004
). Moreover,
it appears that EGF receptor activation in developing mouse pancreas acts to
maintain these undifferentiated nestin-positive precursors at the expense of
differentiated acinar cells (Esni et al.,
2004
). The ability of TGF
to reactivate nestin expression
in mature amylase-positive acinar cells may therefore represent an adult
recapitulation of these embryonic events. In any case, the re-emergence of
nestin expression in mature acinar cells suggests that fully differentiated
pancreatic epithelial cells may act as latent or facultative precursors.
However, unlike transdifferentiation in urodeles, we did not detect completely
undifferentiated intermediate cell types in our system. Rather,
transdifferentiating acinar cells displayed concomitant expression of nestin
and acquisition of a ductal marker (cytokeratins) before the complete loss of
acinar markers. Although the retention or non-retention of multiple lineage
markers may simply be a function of marker protein stability, the rapid
progression and relative lack of proliferation in our system further
distinguish this form of transdifferentiation from that observed during
urodele tail regeneration (Echeverri and
Tanaka, 2002
).
Recently, it has been reported that human ß-cells are capable of
generating new ß-cells through a process of epithelial-to-mesenchymal
transition (Gershengorn et al.,
2004). This event apparently involves ß-cell
dedifferentiation to generate nestin-positive islet precursor cells displaying
mesenchymal features. Under appropriate conditions, these precursors
subsequently redifferentiate, producing new ß-cells. In our system,
however, nestin-positive cells retained expression of epithelial markers, and
maintained an organized epithelial architecture. Although acinar cell
transdifferentiation therefore cannot be considered a formal example of
epithelial-mesenchymal transition, we cannot entirely exclude the possibility
that a transient mesenchymal state is also present in our system.
Acinar cell transdifferentiation and the presence or absence of dedicated stem cells in adult pancreas
Rather than relying exclusively on a dedicated precursor population, adult
pancreatic tissue appears capable of recruiting differentiated cell types as a
source of novel and/or replacement cells. In the case of endocrine pancreas,
recent Cre/lox-based lineage tracing studies have suggested that, both in the
course of normal renewal as well as during accelerated islet neogenesis
following partial pancreatectomy, new ß-cells are generated from a pool
of pre-existing, fully differentiated cells defined by the ability to express
an insulin-CreER transgene (Dor et al.,
2004). The current data support a similar ability for
differentiated acinar cells to assume a precursor function in exocrine
pancreas. Although the mechanism for generation of metaplastic ductal
epithelium may certainly differ from the mechanisms employed for renewal of
normal ductal epithelium, the results suggest that fully differentiated acinar
cells retain a latent precursor potential. This model is consistent with the
view that precursor activity may not necessarily be limited to a discrete
population of undifferentiated, pluripotent cells within a given tissue, but
rather might be considered an inducible biologic function of fully
differentiated cells (Blau et al.,
2001
; Shen et al.,
2000
).
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/16/3767/DC1
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