Differentiation of Pancreatic Cells into Hepatocytes
The phenomenon of transdifferentiation is defined
as the conversion of one differentiated cell type to
another (Tosh and Slack, 2002). Generally, cells that
have the potential to transdifferentiate arise from adjacent
regions in the developing embryo. Therefore,
transdifferentiation between adult cells probably
reflects their close developmental relationship.
Numerous examples of transdifferentiation have been
described in literature (Eguchi and Kodama, 1993).
Two examples are the transdifferentiation of pancreas
to liver (reviewed in Tosh and Slack, 2002; Shen et al.
2003) and the reverse, liver to pancreas conversion
(Horb et al.
, 2003). The liver and pancreas exhibit a
close developmental relationship, as they arise from
the same region of the endoderm (Wells and Melton,
1999). We have developed two different in vitro
approaches for inducing the transdifferentiation of
pancreatic cells to hepatocytes. The conversion of pancreatic
cells to hepatocytes can be induced by culture
of either the pancreatic cell line AR42J (Longnecker et al.
, 1979; Christophe, 1994) or the mouse embryonic
pancreas (Percival and Slack, 1999). The first system,
AR42J cells, is a cell line originally isolated from a
carcinoma of an azaserine-treated rat (Longnecker et al.
, 1979); although a single cell type, they are considered
to be amphicrine in nature, i.e., they possess both
exocrine and neuroendocrine properties (Christophe,
1994). The expression of the exocrine enzyme amylase
can be enhanced by short-term culture with 10nM
dexamethasone (Logsdon et al.
, 1985). The advantage
of the second system for studying transdifferentiation,
the cultured embryonic pancreas system, is that it is
more physiological than the AR42J cell line, which has been around for more than 20 years (Shen et al.
In addition, as the dorsal pancreatic organ grows as a
flattened, branched structure, it is suitable for whole
mount immunostaining. As well as being of interest to
individuals who plan to investigate the transdifferentiation
of pancreas to liver, the system is also relevant
to those working on normal pancreas development.
This article describes the use of AR42J cells and mouse
embryonic pancreas as models for the transdifferentiation
of pancreas to liver.
A. Models for Differentiation of Pancreatic
Cells to Hepatocytes
Several protocols have been produced for inducing
the in vivo
appearance of hepatocytes in the pancreas.
For example, administration of a methionine-deficient
diet and exposure to a carcinogen (Scarpelli and Rao,
1981) can induce hepatocytes in the pancreas of
hamsters. However, one of the most efficient means
of converting pancreas to liver is to feed rats a
copper-deficient diet in combination with a copper
chelator, Trien (Rao et al.
, 1988). After 7-9 weeks of
copper deficiency, the animals are returned to their
normal diet and hepatocytes begin to appear soon
afterwards. Hepatocytes in the pancreas express a
range of liver-specific proteins, e.g., albumin, and are
able to respond to xenobiotics (Rao et al.
, 1982, 1988).
One drawback to in vivo
studies is that it is
difficult to study individual changes at the cellular or
molecular levels. An alternative approach is to use in vitro
models, e.g., AR42J cells. The hepatocytes that
are induced to differentiate from pancreatic AR42J
Cells express many of the proteins that are found in
normal liver, e.g., albumin, glucose-6-phosphatase,
transferrin, transthyretin, and proteins involved in detoxification (e.g., UDP-glucuronosyltransferases,
CYP family) (Shen et al.
, 2000; Tosh et al.
, 2002). This
system offers the ability to generate hepatocytes that
express a whole range of liver proteins while at the same
time avoiding the necessity to isolate hepatocytes from in vivo
. It also permits the opportunity to study factors
for inducing the conversion of one cell type to another.
The in vitro
culture of mouse embryonic pancreas is
particularly suitable for whole mount immunostaining.
This in turn provides a three-dimensional visualisation
of the cell arrangements (Percival and Slack,
1999; Horb and Slack, 2000). The current system offers
a relatively simple approach and depends on the presence
of a substrate (in this case fibronectin), orientation
of the cut pancreas, and a serum-rich medium.
B. Induction of Transdifferentiation
Both AR42J cells and embryonic pancreas can be
induced to transdifferentiate to hepatocytes by exposure
to the glucocorticoid dexamethasone. We find
dexamethasone to be sufficient. To unambiguously
demonstrate the conversion of pancreatic cells to
hepatocytes, a number of criteria have to be fulfilled.
These include (1) the phenotypic characterisation of
the parent cells (i.e., pancreatic cells) and the descendants
(the liver cells) and (2) the lineage relationship
between the ancestor and the descendant. Characterization
of the phenotypes can be morphological and
biochemical and/or molecular. In the case of AR42J
cells, they exhibit an exocrine phenotype. Therefore
the cells can be characterised with markers of digestive
enzymes, e.g., amylase. Because the embryonic
pancreas contains both exocrine and endocrine cell
types, it is possible, with the correct combination of
antibodies, to immunostain for at least three cell types
(exocrine cells, glucagon-secreting α cells, and insulinsecreting
β cells). In contrast, hepatocytes exhibit a vast
array of proteins, including albumin, transferrin, and
transthyretin so it is easy to determine the expression
of the descendant. The second criterion (to demonstrate
the ancestor-descendant relationship) can be
satisfied by using the Cre-lox system in vivo
2000) or by lineage labelling in vitro
, e.g., using green
fluorescent protein (GFP) (Shen et al.
Dexamethasone can be replaced by the naturally
occurring glucocorticoid cortisol to induce the conversion
of AR42J cells to hepatocytes. To determine
whether the effect of the glucocorticoid is specific, the
cells can be exposed to RU486, the glucocorticoid
receptor antagonist, prior to the addition of dexamethasone
or cortisol. Furthermore, the number of
AR42J cells that will transdifferentiate can be enhanced
by the culture of dexamethasone in combination with oncostatin M. Oncostatin M is a member of the IL-6
family of interleukins and has been shown to enhance
the maturation of embryonic liver (Kamiya et al.
II. MATERIALS AND
Dexamethasone (D1756) and cortisol are from
Sigma Chemical Co (St. Louis, MO). RU-486 (Mifepristone)
is from Biomol Research Laboratories, Inc. (Plymouth,
PA). Recombinant human oncostatin M is from
R&D System Inc. (Minneapolis, MN). Dulbecco's
minimum essential medium (DMEM; D5546), basal
medium Eagle (BME, B1522), minimum essential
medium Eagle (MEM Eagle, M5775), penicillin-streptomycin
solution (10,000U/ml/10-mg/ml stock,
P4333), and L-glutamine (G7153) are from Sigma
(Poole, Dorset, UK). Trypsin-EDTA solution (25300-
054), gentamycin (15710-049), and fetal bovine serum
(FBS) (10106-169) are all from Invitrogen (Paisley, UK).
Tissue culture 35-mm plastic dishes are from Fischer
and 22 × 22-mm (MIC 3114) glass coverslips are from
Scientific Laboratory Supplies (Nottingham, UK).
Blocking reagent (1 096 176) is from Roche (Welwyn
Garden City, UK).
Phosphate-buffered saline (PBS) tablets are supplied
by Oxoid Ltd. (Basingstoke, UK). MilliQ-filtered
O is sterilised by autoclaving. Dissecting instruments,
including small scissors, large scissors, tungsten
wire needle (Goodfellow Metals, Cambridge, UK)
in a glass capillary tube, and two pairs of forceps
(Dumont No. 5, Sigma F6521), are required.
A. Cell Lines and Culture Conditions
Transdifferentiation of AR42J Cells to Hepatocytes
AR42J cells can be obtained as a frozen aliquot or
growing culture from the ECACC (93100618) or ATCC
(CRL-1492). AR42J-B13 cells (kindly provided by Dr.
Itaru Kojima, Japan) are a subclone of the parent line
AR42J. The subclone was isolated on the basis of an
increased tendency to convert to β cells (Mashima et al.
, 1996). Either cell type can be induced to transdifferentiate
to hepatocytes, the difference being that the
AR42J-B13 subclone transdifferentiates more readily
than the parent cell line (Shen et al.
, 2000). Cells are
maintained in Dulbecco's modified Eagle's medium
containing penicillin, streptomycin, and 10% fetal bovine serum. Dex (1 µM) is added as a solution in
ethanol, and medium is changed every 1-2 days.
RU486 is added at a concentration of 2.5 µM
, with the
treatment commencing 1 h before addition of the Dex.
Oncostatin M is added as a solution in phosphatebuffered
saline containing 0.1% bovine serum albumin
at a final concentration of 10ng/ml together with
B. Other Procedures
Immunofluorescence Analysis and Antisera
- For immunofluorescent staining, culture AR42JB13
cells on noncoated glass coverslips, rinse with PBS,
and fix with 4% paraformaldehyde in PBS for 30rain.
Immunostain the cells on a coverslip as described later
and mount on a microscope slide in a suitable mounting
media such as gelvatol.
- Permeabilise with 0.1% (v/v) Triton X-100 in PBS
for 30 rain.
- Incubate the coverslips in 2% blocking buffer
(Roche) and 0.1% (v/v) Triton X-100, and then incubate
sequentially with primary and secondary antibodies.
Dilute and obtain the antibodies as follows:
rabbit polyclonal antiamylase (A8273; 1/300) and
rabbit polyclonal antialbumin (1/500; A0433), both
from Sigma Chemical Co., and rabbit polyclonal antitransferrin
(A0061, 1/200) and rabbit polyclonal antitransthyretin
(A0062, 1/100), both from DAKO (Ely,
Cambridge). The goat polyclonal antirabbit IgG FITC
conjugate (FI-1000; 1/150) is from Vector Laboratories
|FIGURE 1 Immunostaining for albumin in control (A) and dexamethasone-treated AR42J-B13 cells (B).
C. Isolation of Mouse Embryonic Pancreas
- Produce gelvatol medium by dissolving 20g of
polyvinyl alcohol in 80ml of 10mM Tris, pH 8.6,
along with 0.2% NaN3.
- In a separate tube, mix 3g of n-propyl gallate and
50ml glycerol until clear. This will take 2-3 days at
room temperature with rotating.
- When both solutions are dissolved, mix the two.
Occasionally lumps are present, which can be
removed by centrifugation at 2500rpm for 15 min.
- Pour off the supernatant into a new tube, leaving
the lumps at the bottom of the old tube.
- This tube can now be wrapped in foil and stored
This procedure is modified from Percival and Slack
(1999) and from Horb and Slack (2000).
Embryonic Culture of Mouse Pancreatic Buds
Isolate dorsal pancreatic buds from 11.5-day mouse
embryos as described later. Following isolation, dissect
the pancreatic buds out in minimum essential medium
with Hank's salts (Hank's medium) containing 10%
glutamine, and 50µg/ml gentamycin.
Culture the buds on fibronectin-coated coverslips in
medium containing basal medium Eagle with Earle's
salts (Earle's medium), 2 mM glutamine, 50 gg/ml
gentamycin, and 20% FBS. Change the medium daily
for up to 6 days.
Preparation of Fibronectin-Coated Coverslips
Fibronectin comes as a lyophilised powder (Invitrogen,
Cat. No. 33010-018).
Preparation of Embryos
- Add 1 ml of sterilised water to a 1-mg vial of
fibronectin and dissolve (1 mg/ml final concentration).
- Aliquot 50µl into 1-ml Eppendorfs and freeze at
- Prepare glass coverslips by baking for at least 3 h at
- Prior to adding fibronectin, coat the coverslips with
2% 3-aminoporpyltriethoxysilane (APTS) to
enhance attachment of the bud to the coverslip.
- To coat the coverslip with fibronectin, take one tube
of the frozen 50 µl fibronectin and add 950 µl of sterilised
water (make sure it is well dissolved).
- In the tissue culture hood, pipette 50µl of solution
onto a sterilised coverslip that has been placed in a
35-mm culture dish. The final concentration of
fibronectin is 50µg/ml.
- Allow the fibronectin to dry on the coverslips. This
takes 2-3 h.
Mouse embryos are generated by timed matings.
The day of the vaginal plug is taken as 0.5. For the
purpose of the present study, we use 11.5-day embryos.
Mouse Embryonic Dissection and Culture
- Kill mouse by cervical dislocation.
- Test reflexes generally by gripping the paw. If
reflexes are absent, then proceed.
- Soak the fur in 70% ethanol. This is generally a
source of infection.
- Using blunt forceps and scissors, cut open the
abdomen (first layer) by a small incision and then tear
this back with the fingers and expose the peritoneal
- Cut the layer with the sharp scissors and forceps
and expose one of the uterine horns by displacing the
abdominal contents and fatty tissue to the side.
- Find the furthest embryo from the base of the
uterus in either horn and remove the whole string of
embryos into a 90-mm petri dish containing ice-cold
- Separate the embryos from each other using
blunt forceps and scissors; alternatively, simply hold
the embryo and uterus with blunt forceps in one hand
and squeeze gently until the embryo pops out of the
uterus. Place embryos in a clean petri dish of PBS.
- Dissect the embryos from the sacs and remove
their heads. Place in a new petri dish containing
- Dissect open the embryo and locate the stomach.
At the lower end, just as the stomach empties into the
intestine, there is a tissue lying over the surface of the
organ. This is the dorsal embryonic bud. Separate
the stomach (along with the dorsal pancreas) from the
intestine and liver using the needle as a knife. Always
remember to cut away from the tissue that you require
to avoid damaging the delicate tissue. Finally, with the
needle, prise the pancreas away from the stomach and
place in a fresh dish of Hank's medium.
- To culture the pancreatic bud, first place a
cloning ring on top of the fibronectin-coated region of
the coverslip. Fibronectin is visible as a dried "ring"
on the coverslip so it is easy to locate. Add 2.5ml of
Earle's culture medium. Initially add some medium
dropwise to the cloning ring but avoid bubbles, as this
makes it difficult for the pancreas to settle and attach
to the substratum.
- For each pancreas culture, suck up tissue into a
pipette and lower onto medium in the cloning ring.
Although two to three pancreatic buds can be cultured
within a single cloning ring, they sometimes grow and
overlap so it is best to culture only one per dish.
- Centre the pancreas with the tungsten needle
and make sure that the cut surface of the pancreas bud
is face down. It is important that the culture is placed
in the centre, as occasionally the cloning ring can move
slightly and could crush the bud, especially if it is too
close to the edge of the cloning ring. Also, ensuring
that the bud is placed cut surface down will increase
the chances of the bud sticking to the fibronectin
- Place the dish in the incubator at 37°C and
- The following day, check the cultures under an
inverted microscope. Generally, allow 24h before
changing the medium. Remove the cloning rings from
the coverslips with forceps and then change the
medium to fresh medium. Repeat medium changes
every 1-2 days (Fig. 2).
|FIGURE 2 Immunostaining for amylase, insulin, and glucagon in normal pancreatic buds. Pancreatic buds
were cultured for 7 days and then immunostained for amylase (A), insulin, (B), and glucagon (C).
The cultured pancreatic buds generally flatten out
onto the substratum over the first 1-2 days, and mesenchymal
cells spread rapidly out of the explant to
form a monolayer of cells surrounding the epithelia in
the centre. On the second or third day, branches begin
to appear in the epithelium. Scattered cells expressing
insulin or glucagon become evident during the first 3
days in culture. Over the next 3 days, the epithelium
becomes an extended branched structure radiating
from the original centre, and the development of
exocrine cells can be recognized. Insulin cells become
more numerous and strongly stained, and some isletlike
architecture can be seen from day 6. These time courses show that the cell differentiation in the cultures
resembles quite closely what occurs in vivo
although it is delayed about I day over a 4-day culture
Induction of Transdifferentiation
To induce transdifferentiation of pancreatic cells to
hepatocytes, add 1 µM dexamethasone. This should be
added at 1- to 2-day intervals from the 1 mM
is kept at-20°C. Dexamethasone can be added to the
medium prior to pipetting onto the dishes or directly
to the dish. Add an equivalent volume of ethanol to
The embryonic buds can be analysed by immunofluorescent
analysis, but the fixation and permeabilisation
conditions are different from those for AR42J
D. Immunofluorescence Analysis of
- For immunofluorescent staining, fix the pancreata
in MEMFA (10% formaldehyde, 0.1M Mops, pH
7.4, 2 mM, EGTA, 1 mM MgSO4) for 30-45 min at room
- Wash in PBS; they can be stored in PBS at 4°C for
up to a few days.
- Prior to immunostaining, treat the cultures with
1% Triton X-100 in PBS to permeabilise and then block
in 2% blocking buffer (Roche), which contains 0.1%
- The buds can then be incubated sequentially
with primary and secondary antibodies. We can
perform triple labelling for the detection of amylase
(FITC), insulin (TRITC), and glucagon (AMCA). Dilute
the primary antibodies in blocking buffer and apply to
the coverslip overnight at 4°C. Dilute and obtain the
antibodies as follows: rabbit polyclonal antiamylase
(1/300, Sigma A8273), guinea pig polyclonal antiinsulin
(1/300, Sigma, I6163), mouse monoclonal antiglucagon (1:100, Sigma G2654), goat polyclonal
antirabbit IgG FITC conjugate (1/100; Vector Laboratories
FI-1000), rabbit polyclonal antiguinea pig IgG
TRITC conjugate (1/300; Sigma T7153), and horse antimouse
IgG AMCA conjugate (1/100; Vector Laboratories
CI-2000). The following day, wash the coverslips
three times (15 min each) and apply the secondary antibody
and leave on for 3 h. On the last day (day 4), wash
the coverslip again (three times 15min each) and
mount the coverslip in an appropriate medium, e.g.,
gelvatol or mobiol. View specimens under a fluorescent
microscope. After induction of transdifferentiation
with dexamethasone, the hepatocytes can be
detected in buds by using the liver antibodies
described in Section III,B.
For confocal imaging, we use a Zeiss LSM 510 confocal
system on an inverted Zeiss fluorescent microscope
fitted with a ×40/NA 1.30 or ×63/NA 1.40 oil
immersion objective (Zeiss, Welwyn Garden City, UK).
Alternatively, we use a Leica DMRB microscope fitted
with a digital camera. Generally, when two or more
antibodies are visualized through different fluorescent
channels in the same specimen, the initial black-andwhite
CCD images are coloured and then recombined
to form a single multicoloured image.
The differentiation of pancreatic AR42J cells to
hepatocytes results in a marked morphological
change. The cells become flattened and enlarged.
These changes occur with 5 days of dexamethasone
treatment. It is therefore possible to check the differentiation
by noting the number and degree of flattening
- For the best results with the embryonic pancreatic
cultures, ensure that the bud is placed cut surface
down and in the centre of the cloning ring. If not, there
is a chance that the cloning ring will move when the
incubator door is closed, squashing the bud.
- When immunostaining for liver proteins, do not
be tempted to use serum as a blocking agent. Although
good for conventional immunostaining, it is not suitable
for some of the liver markers, which are serum proteins
normally secreted from the organ, e.g., albumin.
- Prior to inducing transdifferentiation, inoculate
the AR42J cells at low density (e.g., we routinely inoculate
at 10-15%). These cells have a high rate of cell
division and although dexamethasone inhibits cell
division, you may end up with too confluent a dish for
This work was supported by the Medical Research
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