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., 2000). 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 1 µM 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 (Herrera, 2000) or by lineage labelling in vitro, e.g., using green fluorescent protein (GFP) (Shen et al., 2000).
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., 1999).
II. MATERIALS AND INSTRUMENTATION
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 H2O 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 1 µM Dex.
B. Other Procedures
Immunofluorescence Analysis and Antisera
C. Isolation of Mouse Embryonic Pancreas
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% FBS, 2mM 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
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
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 period.
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 stock that 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 control dishes.
The embryonic buds can be analysed by immunofluorescent analysis, but the fixation and permeabilisation conditions are different from those for AR42J cells.
D. Immunofluorescence Analysis of Embryonic Pancreas
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 of cells.
This work was supported by the Medical Research Council.
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