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Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, Bode JG, Bolleyn J, Borner C, Böttger J, Braeuning A, Budinsky RA, Burkhardt B, Cameron NR, Camussi G, Cho CS, Choi YJ, Craig Rowlands J, Dahmen U, Damm G, Dirsch O, Donato MT, Dong J, Dooley S, Drasdo D, Eakins R, Ferreira KS, Fonsato V, Fraczek J, Gebhardt R, Gibson A, Glanemann M, Goldring CE, Gómez-Lechón MJ, Groothuis GM, Gustavsson L, Guyot C, Hallifax D, Hammad S, Hayward A, Häussinger D, Hellerbrand C, Hewitt P, Hoehme S, Holzhütter HG, Houston JB, Hrach J, Ito K, Jaeschke H, Keitel V, Kelm JM, Kevin Park B, Kordes C, Kullak-Ublick GA, LeCluyse EL, Lu P, Luebke-Wheeler J, Lutz A, Maltman DJ, Matz-Soja M, McMullen P, Merfort I, Messner S, Meyer C, Mwinyi J, Naisbitt DJ, Nussler AK, Olinga P, Pampaloni F, Pi J, Pluta L, Przyborski SA, Ramachandran A, Rogiers V, Rowe C, Schelcher C, Schmich K, Schwarz M, Singh B, Stelzer EH, Stieger B, Stöber R, Sugiyama Y, Tetta C, Thasler WE, Vanhaecke T, Vinken M, Weiss TS, Widera A, Woods CG, Xu JJ, Yarborough KM, Hengstler JG - Arch. Toxicol. (2013)

Bottom Line: When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes.One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation.Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

View Article: PubMed Central - PubMed

Affiliation: Leibniz Research Centre for Working Environment and Human Factors (IFADO), 44139, Dortmund, Germany.

ABSTRACT
This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

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Influence of hepatocyte culture conditions on signaling and phenotype. When hepatocytes are isolated from the organ and cultured on stiff and dry collagen (“collagen monolayers”), at least two signaling pathways (ERK and Akt) are activated. The activation process involves focal adhesion kinase (FAK) and src although the exact mechanisms initiating this process are still unknown. Signaling via the Ras/Raf/ERK pathway (“MAP kinase”) causes dedifferentiation and epithelial to mesenchymal transition (EMT). Activation of the PI3K/Akt pathway causes resistance to apoptosis. Both pathways are strongly activated in collagen monolayers but much less in collagen sandwich cultures (Godoy et al. 2009)
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Fig28: Influence of hepatocyte culture conditions on signaling and phenotype. When hepatocytes are isolated from the organ and cultured on stiff and dry collagen (“collagen monolayers”), at least two signaling pathways (ERK and Akt) are activated. The activation process involves focal adhesion kinase (FAK) and src although the exact mechanisms initiating this process are still unknown. Signaling via the Ras/Raf/ERK pathway (“MAP kinase”) causes dedifferentiation and epithelial to mesenchymal transition (EMT). Activation of the PI3K/Akt pathway causes resistance to apoptosis. Both pathways are strongly activated in collagen monolayers but much less in collagen sandwich cultures (Godoy et al. 2009)

Mentions: With the advent of gene expression profiling, it would be of interest to use cultured hepatocytes also for toxicogenomic studies (see section “Toxicogenomics, a rapidly evolving subdiscipline of toxicology”). Currently, this is hampered by the fact that cultured hepatocyte cultures show major alterations in gene expression compared to the in vivo situation. When placed into culture, more than 3,000 genes are up- or downregulated by at least threefold. Principle component analysis (PCA) of gene array data demonstrated that most up- or downregulations occur during the first 24 h of culture, followed by a period with much smaller expression alterations (Fig. S4(a); see 10.1007/s00204-013-1078-5) (Zellmer et al. 2010). This PCA also included liver tissue of mice (L) which shows only relatively small differences compared to the freshly isolated hepatocytes (H). In a next step, only genes altered during culture were included into the PCA (Fig. S4(b); see 10.1007/s00204-013-1078-5). This analysis illustrates that the three mice (indicated by blue pink and red color) can be differentiated along axis 1. More importantly, axis 2 comprises genes whose expression was altered during the culture period (Fig. S4(b); see 10.1007/s00204-013-1078-5). To understand the mechanism responsible for the observed time-dependent gene expression alterations, the genes associated with axis 2 (Fig. S4(b); see 10.1007/s00204-013-1078-5) were analyzed for over-represented transcription factor binding sites (Zellmer et al. 2010). After adjustment for multiple testing, ETF, Elk-1 and NF-Y were identified as over-represented. An important factor that these transcription factors have in common is that they are influenced by MAP kinase signaling. This led to the hypothesis that MAP kinase signaling might be activated in cultured hepatocytes compared to the in vivo situation. Indeed, phosphorylated ERK1/2 was clearly increased in cultured hepatocytes compared to liver tissue (Fig. 27; Godoy et al. 2009). This increase in phosphorylated ERK1/2 was already present in freshly isolated hepatocytes, indicating that activation of MAP kinase signaling is not only a consequence of the culture conditions (see 24–72 h in culture in Fig. 27) but also of the isolation stress. Similar to ERK1/2, Akt phosphorylation was also increased. The degree of Akt and ERK1/2 activation strongly depends on the culture conditions with much stronger phosphorylation seen in hepatocytes cultured as monolayers than in sandwich culture (Fig. 27). Both activation of the signaling via ERK1/2 and Akt have major consequences on the hepatocyte phenotype. Activation of ERK1/2, e.g. by expression of a constitutively active Ras, but not constitutively active Akt leads to hepatocyte dedifferentiation and to features of epithelial to mesenchymal transition—EMT (Fig. 28; Godoy et al. 2009). Activation of Akt causes resistance of apoptosis by antagonizing activation of p38. This has been shown by inhibitor experiments in hepatocyte monolayers where inhibition of PI-3 kinase restored apoptosis sensitivity (Godoy et al. 2009). Similarly, over-expression of a constitutively active Akt in sandwich cultures (representing conditions without Akt activation and apoptosis resistance) induced resistance to apoptosis. Therefore, activation of Akt as a consequence of the culture conditions is of relevance for toxicity experiments and may lead to underestimation of in vivo toxicity. Both activation of ERK1/2 and Akt and its biological consequences (EMT and apoptosis resistance) are reversible. Upon harvesting hepatocytes from collagen monolayers and re-plating them into sandwich cultures, the degree of ERK1/2 and Akt phosphorylation decreased (Godoy et al. 2009). This was accompanied by a re-establishment of the differentiated phenotype, a decrease in EMT markers and by re-establishment of apoptosis sensitivity.Fig. 27


Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, Bode JG, Bolleyn J, Borner C, Böttger J, Braeuning A, Budinsky RA, Burkhardt B, Cameron NR, Camussi G, Cho CS, Choi YJ, Craig Rowlands J, Dahmen U, Damm G, Dirsch O, Donato MT, Dong J, Dooley S, Drasdo D, Eakins R, Ferreira KS, Fonsato V, Fraczek J, Gebhardt R, Gibson A, Glanemann M, Goldring CE, Gómez-Lechón MJ, Groothuis GM, Gustavsson L, Guyot C, Hallifax D, Hammad S, Hayward A, Häussinger D, Hellerbrand C, Hewitt P, Hoehme S, Holzhütter HG, Houston JB, Hrach J, Ito K, Jaeschke H, Keitel V, Kelm JM, Kevin Park B, Kordes C, Kullak-Ublick GA, LeCluyse EL, Lu P, Luebke-Wheeler J, Lutz A, Maltman DJ, Matz-Soja M, McMullen P, Merfort I, Messner S, Meyer C, Mwinyi J, Naisbitt DJ, Nussler AK, Olinga P, Pampaloni F, Pi J, Pluta L, Przyborski SA, Ramachandran A, Rogiers V, Rowe C, Schelcher C, Schmich K, Schwarz M, Singh B, Stelzer EH, Stieger B, Stöber R, Sugiyama Y, Tetta C, Thasler WE, Vanhaecke T, Vinken M, Weiss TS, Widera A, Woods CG, Xu JJ, Yarborough KM, Hengstler JG - Arch. Toxicol. (2013)

Influence of hepatocyte culture conditions on signaling and phenotype. When hepatocytes are isolated from the organ and cultured on stiff and dry collagen (“collagen monolayers”), at least two signaling pathways (ERK and Akt) are activated. The activation process involves focal adhesion kinase (FAK) and src although the exact mechanisms initiating this process are still unknown. Signaling via the Ras/Raf/ERK pathway (“MAP kinase”) causes dedifferentiation and epithelial to mesenchymal transition (EMT). Activation of the PI3K/Akt pathway causes resistance to apoptosis. Both pathways are strongly activated in collagen monolayers but much less in collagen sandwich cultures (Godoy et al. 2009)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3753504&req=5

Fig28: Influence of hepatocyte culture conditions on signaling and phenotype. When hepatocytes are isolated from the organ and cultured on stiff and dry collagen (“collagen monolayers”), at least two signaling pathways (ERK and Akt) are activated. The activation process involves focal adhesion kinase (FAK) and src although the exact mechanisms initiating this process are still unknown. Signaling via the Ras/Raf/ERK pathway (“MAP kinase”) causes dedifferentiation and epithelial to mesenchymal transition (EMT). Activation of the PI3K/Akt pathway causes resistance to apoptosis. Both pathways are strongly activated in collagen monolayers but much less in collagen sandwich cultures (Godoy et al. 2009)
Mentions: With the advent of gene expression profiling, it would be of interest to use cultured hepatocytes also for toxicogenomic studies (see section “Toxicogenomics, a rapidly evolving subdiscipline of toxicology”). Currently, this is hampered by the fact that cultured hepatocyte cultures show major alterations in gene expression compared to the in vivo situation. When placed into culture, more than 3,000 genes are up- or downregulated by at least threefold. Principle component analysis (PCA) of gene array data demonstrated that most up- or downregulations occur during the first 24 h of culture, followed by a period with much smaller expression alterations (Fig. S4(a); see 10.1007/s00204-013-1078-5) (Zellmer et al. 2010). This PCA also included liver tissue of mice (L) which shows only relatively small differences compared to the freshly isolated hepatocytes (H). In a next step, only genes altered during culture were included into the PCA (Fig. S4(b); see 10.1007/s00204-013-1078-5). This analysis illustrates that the three mice (indicated by blue pink and red color) can be differentiated along axis 1. More importantly, axis 2 comprises genes whose expression was altered during the culture period (Fig. S4(b); see 10.1007/s00204-013-1078-5). To understand the mechanism responsible for the observed time-dependent gene expression alterations, the genes associated with axis 2 (Fig. S4(b); see 10.1007/s00204-013-1078-5) were analyzed for over-represented transcription factor binding sites (Zellmer et al. 2010). After adjustment for multiple testing, ETF, Elk-1 and NF-Y were identified as over-represented. An important factor that these transcription factors have in common is that they are influenced by MAP kinase signaling. This led to the hypothesis that MAP kinase signaling might be activated in cultured hepatocytes compared to the in vivo situation. Indeed, phosphorylated ERK1/2 was clearly increased in cultured hepatocytes compared to liver tissue (Fig. 27; Godoy et al. 2009). This increase in phosphorylated ERK1/2 was already present in freshly isolated hepatocytes, indicating that activation of MAP kinase signaling is not only a consequence of the culture conditions (see 24–72 h in culture in Fig. 27) but also of the isolation stress. Similar to ERK1/2, Akt phosphorylation was also increased. The degree of Akt and ERK1/2 activation strongly depends on the culture conditions with much stronger phosphorylation seen in hepatocytes cultured as monolayers than in sandwich culture (Fig. 27). Both activation of the signaling via ERK1/2 and Akt have major consequences on the hepatocyte phenotype. Activation of ERK1/2, e.g. by expression of a constitutively active Ras, but not constitutively active Akt leads to hepatocyte dedifferentiation and to features of epithelial to mesenchymal transition—EMT (Fig. 28; Godoy et al. 2009). Activation of Akt causes resistance of apoptosis by antagonizing activation of p38. This has been shown by inhibitor experiments in hepatocyte monolayers where inhibition of PI-3 kinase restored apoptosis sensitivity (Godoy et al. 2009). Similarly, over-expression of a constitutively active Akt in sandwich cultures (representing conditions without Akt activation and apoptosis resistance) induced resistance to apoptosis. Therefore, activation of Akt as a consequence of the culture conditions is of relevance for toxicity experiments and may lead to underestimation of in vivo toxicity. Both activation of ERK1/2 and Akt and its biological consequences (EMT and apoptosis resistance) are reversible. Upon harvesting hepatocytes from collagen monolayers and re-plating them into sandwich cultures, the degree of ERK1/2 and Akt phosphorylation decreased (Godoy et al. 2009). This was accompanied by a re-establishment of the differentiated phenotype, a decrease in EMT markers and by re-establishment of apoptosis sensitivity.Fig. 27

Bottom Line: When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes.One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation.Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

View Article: PubMed Central - PubMed

Affiliation: Leibniz Research Centre for Working Environment and Human Factors (IFADO), 44139, Dortmund, Germany.

ABSTRACT
This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

Show MeSH
Related in: MedlinePlus