Maintaining hepatocyte differentiation in vitro through co-culture with hepatic stellate cells
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- Krause, P., Saghatolislam, F., Koenig, S. et al. In Vitro Cell.Dev.Biol.-Animal (2009) 45: 205. doi:10.1007/s11626-008-9166-1
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Primary hepatocytes lose their differentiated functions rapidly when in culture. Our aim was to maintain the differentiated status of hepatocytes in vitro by means of vital hepatic stellate cells (HSCs), their soluble and particulate factors and lipid extracts. Hepatocytes were placed into collagen-coated culture dishes in the presence of HSCs at different ages of pre-culture, with or without direct cell to cell contacts, at different cell ratios and in monoculture with cellular HSC components in place of vital cells. Changes in morphology and enhancement of phosphoenolpyruvate carboxykinase (PCK) activity by glucagon were used to determine the differentiated status of hepatocytes in 2d-short-term culture. HSCs proved able to maintain the differentiated function of hepatocytes in co-culture either by direct cell contacts or via factors derived from HSC-conditioned medium. In comparison, however, without cellular contact to hepatocytes five to ten times as many HSCs were necessary to increase the PCK activity to the same degree as in the presence of intercellular contacts. Whereas stimulation in the presence of HSC/hepatocyte contacts was independent of HSC culture age only quiescent, resting HSCs (precultured for 1–2 d) were able to stimulate hepatocytes significantly via soluble factors. Culturing of hepatocytes with a lipid extract or a particulate fraction from HSCs clearly displayed a very strong beneficial effect on enzyme activity and morphology. HSCs maintain hepatocyte function and structure through preferentially cell-bound signalling and transfer of lipids.
KeywordsHepatic stellate cellsHepatocytesDifferentiationPhosphoenolpyruvate carboxykinaseCo-culture
When in cell culture, primary hepatocytes are known to lose their differentiated phenotype and vitality rapidly on isolation from their native microenvironment (Arterburn et al. 1995; Runge et al. 2000). Therefore, many investigators have been developing sophisticated cell culture systems aiming to maintain the vitality and specific metabolic functions (Elaut et al. 2006). Elaborate culture media and/or the use of complex extracellular matrices (ECM) help to prevent the metabolic deterioration and re-establish hepatic polarity in primary hepatocyte cultures (Enat et al. 1984; Block et al. 1996; Mitaka 1998). More precisely, not only do the culture medium and cell dish surface play a pivotal role in the maintenance of hepatocytes in vitro, heterotypic cell to cell interactions are also imperative to maintain coordinated cell functions in vitro. Initial attempts were made by preserving the morphology and biochemical integrity of hepatocytes in co-culture with extrahepatic cells (e.g. fibroblasts, endothelial cells) (Langenbach et al. 1979; Talamini et al. 1998). However, the co-culture of mature hepatocytes with non-parenchymal (stromal) cells from the liver itself has been shown to be more effective in modulating cell differentiation (Bader et al. 1996; Bhatia et al. 1999; Ries et al. 2000). Various two and three dimensional co-culture models have demonstrated their ability to mimic the liver microenvironment, suggesting that each hepatic non-parenchymal cell population (sinusoidal endothelium, Kupffer cells and hepatic stellate cells (HSCs)) may contribute to the overall beneficial effect on hepatocytes (Morin and Normand 1986; Okamoto et al. 1998; Riccalton-Banks et al. 2003; Thomas et al. 2005; Zinchenko et al. 2006a).
To date, only few studies have focussed on co-culturing hepatocytes with individual non-parenchymal cell fractions. In particular, the exact role of HSCs needs to be elucidated further with respect to their culture age and to signals generated by membrane contact or by cytoplasmic/soluble factors. HSCs constitute a mesenchymal cell type and display unique features with respect to their cellular origin, morphology and function (Suematsu and Aiso 2001). Normal, quiescent HSCs store over 80% of total body vitamin A and maintain vitamin A blood homoeostasis (Sato et al. 2003). HSCs are located between parenchymal cell plates and sinusoidal endothelial cells and extend well-developed, long processes surrounding sinusoids in vivo as pericytes (Senoo 2004). They are the predominant cell type producing ECM components as well as ECM-degrading metalloproteases, indicating that they play a pivotal role in ECM remodelling in both normal and pathological liver conditions (Kmiec 2001).
The central question of our study was as to whether primary, quiescent HSCs maintain the phenotype and gluconeogenic activity of hepatocytes either in a co-culture setting with direct cell to cell contacts, via soluble factors in the culture medium and/or via cellular components such as particulate cell material and lipids.
Materials and Methods
Animals and Reagents.
Male Wistar rats were purchased from Winkelmann (Borchen, Germany) and kept on a 12 h day/night rhythm with a standard rat diet. Collagenase, epidermal growth factor, basic fibroblast growth factor, as well as neonatal calf serum were purchased from Biochrom (Berlin, Germany). Williams Medium E with 5 mM glucose and holo-transferrin were supplied by Applichem (Darmstadt, Germany). Bovine insulin was purchased from Serva (Heidelberg, Germany), inosine triphosphate from Aldrich (Taufkirchen, Germany), pronase from Merck (Darmstadt, Germany), Nycodenz from Nycomed Pharma (Oslo, Norway) and Percoll from Pharmacia (Freiburg, Germany). All further chemicals were supplied by Sigma (Deisenhofen, Germany).
Isolation of hepatocytes and non-parenchymal (stromal) cells.
Hepatocytes were isolated from 250–300 g rats using the collagenase perfusion protocol exactly as described (Ries et al. 2000). Hepatocytes were re-suspended in Williams E medium with 5 mM glucose containing 4% NCS, 0.2% bovine serum albumin, 15 mM HEPES, 0.2 mM ethanolamine, 0.2 mM phosphoethanolamine, 7.5 nM sodium selenite, 0.1 μM dexamethasone and 1 nM insulin. Hepatocytes were plated onto 60-mm dishes which were pre-coated with rat tail collagen (not crosslinked) and dried for 24 h. After 4 h, the medium was changed to the medium mentioned above; serum was omitted from now on. Thereafter, medium was changed daily (2.5 ml/dish). The supernatant of the first centrifugation contained the non-parenchymal cells (NPCs) as a mixture of endothelial, HSCs and Kupffer cells. These cells were washed twice in Williams medium (sedimentation at 200 g for 10 min) and re-suspended in 5 ml medium, of which 150 μl/dish were plated together with the hepatocytes (Ries et al. 2000).
Isolation and culture of stellate cells.
Cells were isolated using a combined two-step pronase/collagenase digestion (Weiner et al. 1992; Pestel et al. 2003). Briefly, the liver from 350–450 g rats was excised during pre-perfusion with Hanks balanced salt solution (HBSS) without Mg2+ and Ca2+ at a flow rate of 7.5 ml/min. Flow rate was then increased to 20 ml/min and perfusions continued ex situ: first with 100 ml HBSS and 0.035% pronase in a non-recirculating manner and then with 100 ml HBSS plus 0.028% collagenase for 20–30 min under recirculation. The soft liver was mechanically dispersed and the crude tissue aggregates slowly stirred in 100 ml HBSS with 100 mg pronase, 2 mg DNase and 25 mg BSA for 10 min under constant readjustment of the pH to 7.4. Cells were filtered through a 60 μm nylon mesh, freed of hepatocytes by centrifugation (50 g for 7 min), pelleted (450 g for 7 min), re-suspended in HBSS plus DNase and mixed with Nycodenz to a final concentration of 8.1% in a total volume of 160 ml. Aliquots of 20 ml were overlaid with 8 ml HBSS in 30 ml Corex tubes and centrifuged for 20 min at 1,400 g. HSC were obtained from the interphase, washed once, re-suspended in Williams medium with 15% foetal calf serum, 0.1 μM dexamethasone, 1 nM insulin and plated into 60-mm dishes coated with native (not crosslinked) collagen for short-term cultures (24–48 h), or with glutaraldehyde-crosslinked collagen for long-term cultures (>2 d). For short-term cultures, foetal calf serum was omitted after the first medium change (3 h) and long-term cultures were freed of serum 24 h before the experiments. HSCs were identified by their characteristic lipid droplet inclusion (light microscopy, autofluorescence) and positive vimentin staining. Purity was determined as being >90%, vitality 80% and the yield was 30–40 million cells/liver. Kupffer cells were not detected (phagocytosis of fluorescent latex beads, ED2 antibody staining). Occasionally, sinusoidal endothelial cells (<5%, detection by the uptake of fluorescent acetylated LDL) adhered to the dish, but these cells were detached after 20 h of culture.
HSC conditioned medium.
HSC (4 × 106) were cultured as described above on dishes coated with glutaraldehyde-crosslinked collagen. Conditioned media were sampled between 24–48 h of the culture and for long-term cultures in the last serum-free 24-h period. They were sterilised by filtration, centrifuged at 10,000 × g for 30 min and used without storing longer than 24 h at 4°C. Cells received 50% CM (as a 1:1 mixture with hepatocyte culture medium) at the 4 and 24 h changes.
Preparation of HSC particulate fraction.
HSC were pre-cultured for 2 or 7–21 d, washed twice with Williams medium, scraped off the dish and were sonicated in 500 μl medium for 20 s at a low frequency. The particulate fraction was pelleted (14,000 rpm, 20 min), washed once and was re-suspended in medium with 10 nM insulin and 0.1 μM dexamethasone. Hepatocytes were supplied with 1.5 ml culture medium and 500 μl particulate fraction (from 2 × 106 HSC/dish) at the first medium change.
Four dishes each of 3 × 106 HSCs or hepatocytes were cultured for 24 h and used for extraction exactly as described previously (Probst et al. 2003). The chloroform phase was transferred into a sterile 50-ml glass medium flask and the solvent was evaporated under N2 in the dark. Lipids were re-dissolved in 24 ml of Williams medium containing 0.1 μM dexamethasone and 1 nM insulin by stirring at 4°C for 6 h. Lipids were used immediately at a 1:2 dilution with culture medium (lipids of 1 × 106 cells/dish).
Experimental design of cell cultures.
Hepatocyte seeding density was 0.5 or 1 × 106 cells/dish. For co-culturing purposes, HSCs were pre-cultured for various lengths of time, hepatocytes were then seeded onto the HSC dish and cultured for 2 d before the induction of phosphoenolpyruvate carboxykinase (PCK) by glucagon. Ring cultures were used to separate the two cell types physically on one dish (Schrode et al. 1990); a 4-mm plastic ring was cut off a 50-ml Falcon tube, sterilised and glued into the centre of the 60-mm dish using collagen. Hepatocytes were always seeded into the outer ring. HSCs were seeded at different densities either into the outer ring (cell contact with hepatocytes) or into the inner circle (no cell contact). The ring was removed during the first medium change (4 h).
Determination of phosphoenolpyruvate carboxykinase activity.
Cell cultures were washed twice and incubated with Williams E-based culture medium without serum and insulin but containing 0.1 μM dexamethasone and 2 mM lactate. After 30 min, the experiment was initiated by adding 10 nM glucagon. The incubation was terminated after 6 h by rinsing the plates once with 0.9% saline solution and subjecting the dishes to liquid nitrogen. Enzyme activity was determined photometrically (Seubert and Huth 1965).
The dissolution of rat tail collagen and its crosslinking with glutaraldehyde were done as described (Ries et al. 2000). Dishes were coated with either native or crosslinked collagen and allowed to dry for 24 h. Cell morphology was assessed by phase contrast light microscopy (LEICA DM IRE2, Bensheim, Germany).
The beneficial stimulatory effects of a HSC particulate fraction and of the HSC lipid extract on PCK activity were paralleled by maintenance of hepatocyte morphology (Fig. 2c, d). Hepatocytes clearly revealed compact and in part confluent clusters of high contrast cells. The cell nuclei and margins were easily detected, as well as many surrounding bile canaliculi.
Over the last 30 yr, a multitude of studies have demonstrated the maintenance of differentiation when hepatocytes were cultured with a variety of hepatic, non-hepatic epithelial or mesenchymal cells (for a review see Bhatia et al. 1999). Cell associated as well as soluble factors (Lu et al. 2005; Zinchenko et al. 2006a, b) were shown to keep the hepatocyte morphologically and functionally differentiated. The hepatocyte is in intricate contact with spiny extensions of the hepatic stellate cell, its nearest cell neighbour. Here, we demonstrate for the first time that primary isolated quiescent HSCs of a young culture age (1 to 2 d) maintain hepatocyte function and structure through cell contact and soluble factors. Our simple co-culture model allowed us to differentiate between these signals and to estimate soluble and cell-bound signal potency by varying the ratio of hepatocytes to HSCs on the dish (Fig. 3). Both soluble and membrane-bound signal transfer were only detectable when HSCs outnumbered hepatocytes. Bound signals were superior or exclusive when HSC numbers dropped below the number of hepatocytes. This mirrors the cell architecture in vivo, in which one HSC roughly spans two to three hepatocytes (Sato et al. 2003). Soluble factors provided by HSC medium, conditioned in the absence of hepatocytes, were effective only when the HSCs were still of a young culture age and thus in a differentiated, quiescent state (Fig. 5). Cell-associated signals maintained both function and structure irrespective of HSC culture age and vitality, since washed HSC particulate material acted as substitute for the intact living HSCs (Fig. 4). One explanation for this effect may be the presentation of cadherins, providing for homoadhesive cell to cell contacts. HSCs switch cadherin expression from the E- to N-type during in vivo and in vitro activation (Lim et al. 2007) and both E- and N-cadherins as well as the truncated T-type have been identified as potent mediators of hepatocyte differentiation (Khetani et al. 2004, 2008).
HSCs express another surface-associated molecule, epimorphin, which induces in vitro differentiation of hepatocytes (Watanabe et al. 1998) and hepatic stem-like cells (Miura et al. 2003). The protein is known to act as a morphogen during tissue restoration in the adult liver (Yoshino et al. 2006).
Hepatocytes as well as hepatoma cells preserve/regain functionality and structure when cultured with vitamin A (Falasca et al. 1999; Alisi et al. 2003; Bertagnolo et al. 2003). The commercial culture medium used here contains approximately 0.3 μM vitamin A. Any further addition of vitamin A in different stereoforms of retinol or retinoic acid (dissolved in ethanol or dimethylsulfoxide or alternatively bound to albumin) did not enhance differentiation in our pure hepatocyte cultures (results not shown). However, lipids from HSCs cultured for 1 d, extracted with chloroform and bound to albumin, not only maintained hepatocyte function but also preserved cell architecture and polarity very well with the expression of multiple bile canaliculi (Fig. 2d). Nonetheless, it should well be considered that the active component in the lipid extract was not vitamin A alone, since the lipid droplets of HSCs also contain triglycerides, cholesterol and phospholipids (Yamada et al. 1987; Moriwaki et al. 1988). The antiproliferative and differentiative action of vitamin A has been well investigated (Fields et al. 2007); more recently unsaturated fatty acids have been recognised as important signals in diverse processes such as differentiation, development and proliferation (Tontonoz and Spiegelman 2008; Edwards and O’Flaherty 2008). The question remains as to how lipids and especially vitamin A from the intracellular HSC stores reach the neighbouring hepatocytes. Sauvant et al. used primary cultures of HSCs (Sauvant et al. 2001), demonstrating that retinol mobilisation did not require the synthesis of and binding to retinol-binding protein (Kawaguchi et al. 2007). Instead, their data support the idea of direct retinol transfer from the HSCs to hepatocytes by the movement of free retinol in phospholipid bilayer membranes with an increased transfer when direct cell contacts between the two cell types were established.
In conclusion, we show that quiescent HSCs maintain hepatocyte function and structure by multiple signals, preferentially cell bound. The results point to non-soluble membraneous ligand contacts as well as to the direct intercellular transfer of lipids as key factors in the maintenance of hepatocyte differentiation.
The authors would like to express their thanks to Andrew Entwistle for his critical review of this manuscript.
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