Arch Toxicol. 2016 Oct;90(10):2513-29. doi: 10.1007/s00204-016-1761-4. Epub 2016 Jun 23.

Gene network activity in cultivated primary hepatocytes is highly similar to diseased mammalian liver tissue.

Godoy P^1,², Widera A³, Schmidt-Heck W⁴, Campos G³, Meyer C⁵, Cadenas C³, Reif R³, Stöber R³, Hammad S^3,^5,⁶, Pütter L³, Gianmoena K³, Marchan R³, Ghallab A^3,⁶, Edlund K³, Nüssler A⁷, Thasler WE⁸, Damm G⁹, Seehofer D⁹, Weiss TS¹⁰, Dirsch O¹¹, Dahmen U¹², Gebhardt R¹³, Chaudhari U¹⁴, Meganathan K^14,¹⁵, Sachinidis A¹⁴, Kelm J¹⁶, Hofmann U¹⁷, Zahedi RP¹⁸, Guthke R⁴, Blüthgen N¹⁹, Dooley S⁵, Hengstler JG²⁰.

Author information

1: IfADo-Leibniz Research Centre for Working Environment and Human Factors, Technical University of Dortmund, Ardeystrasse 67, 44139, Dortmund, Germany. godoy@ifado.de.
2: Facultad de Ciencias Biológicas, Departamento de Fisiología, Universidad de Concepción, Concepción, Chile. godoy@ifado.de.
3: IfADo-Leibniz Research Centre for Working Environment and Human Factors, Technical University of Dortmund, Ardeystrasse 67, 44139, Dortmund, Germany.
4: Leibniz Institute for Natural Product Research and Infection Biology eV-Hans-Knöll Institute, Jena, Germany.
5: Molecular Alcohol Research in Gastroenterology, Department of Medicine II, Faculty of Medicine at Mannheim, University of Heidelberg, Mannheim, Germany.
6: Department of Forensic and Veterinary Toxicology, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt.
7: BG Trauma Center, Siegfrid Weller Insitut, Eberhard Karls University Tübingen, Tübingen, Germany.
8: Center for Liver Cell Research, Department of General, Visceral, Transplantation, Vascular and Thorax Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich, Germany.
9: Department of General, Visceral and Transplantation Surgery, Charité University Medicine Berlin, Berlin, Germany.
10: Center for Liver Cell Research, University Children Hospital (KUNO), Regensburg University Hospital, Regensburg, Germany.
11: Institute of Pathology, Friedrich-Schiller-University of Jena, Jena, Germany.
12: Experimental Transplantation Surgery, Department of General, Visceral and Vascular Surgery, University Hospital Jena, Friedrich-Schiller-University of Jena, Jena, Germany.
13: Institute of Biochemistry, Faculty of Medicine, University of Leipzig, Leipzig, Germany.
14: Institute of Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany.
15: Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO, USA.
16: InSphero AG, Wagistrasse 27, 8952, Schlieren, Switzerland.
17: Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, University of Tuebingen, Auerbachstrasse 112, 70376, Stuttgart, Germany.
18: Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund, Germany.
19: Institute of Pathology, Charité-Universitätsmedizin Berlin, Berlin, Germany.
20: IfADo-Leibniz Research Centre for Working Environment and Human Factors, Technical University of Dortmund, Ardeystrasse 67, 44139, Dortmund, Germany. hengstler@ifado.de.

Abstract

It is well known that isolation and cultivation of primary hepatocytes cause major gene expression alterations. In the present genome-wide, time-resolved study of cultivated human and mouse hepatocytes, we made the observation that expression changes in culture strongly resemble alterations in liver diseases. Hepatocytes of both species were cultivated in collagen sandwich and in monolayer conditions. Genome-wide data were also obtained from human NAFLD, cirrhosis, HCC and hepatitis B virus-infected tissue as well as mouse livers after partial hepatectomy, CCl4 intoxication, obesity, HCC and LPS. A strong similarity between cultivation and disease-induced expression alterations was observed. For example, expression changes in hepatocytes induced by 1-day cultivation and 1-day CCl4 exposure in vivo correlated with R = 0.615 (p < 0.001). Interspecies comparison identified predominantly similar responses in human and mouse hepatocytes but also a set of genes that responded differently. Unsupervised clustering of altered genes identified three main clusters: (1) downregulated genes corresponding to mature liver functions, (2) upregulation of an inflammation/RNA processing cluster and (3) upregulated migration/cell cycle-associated genes. Gene regulatory network analysis highlights overrepresented and deregulated HNF4 and CAR (Cluster 1), Krüppel-like factors MafF and ELK1 (Cluster 2) as well as ETF (Cluster 3) among the interspecies conserved key regulators of expression changes. Interventions ameliorating but not abrogating cultivation-induced responses include removal of non-parenchymal cells, generation of the hepatocytes' own matrix in spheroids, supplementation with bile salts and siRNA-mediated suppression of key transcription factors. In conclusion, this study shows that gene regulatory network alterations of cultivated hepatocytes resemble those of inflammatory liver diseases and should therefore be considered and exploited as disease models.

KEYWORDS:

Bioinformatics; Differentiation; Gene arrays; Inflammation; Metabolism

PMID:: 27339419
PMCID:: PMC5043005
DOI:: 10.1007/s00204-016-1761-4

[Indexed for MEDLINE]

Free PMC Article

Images from this publication.See all images (8)Free text

Fig. 1

Transcriptional alterations induced by cultivation of primary hepatocytes. a, b Principal component analysis representing the 1000 genes with highest variance in primary mouse (a) and human (b) hepatocytes. The top two principal components (PC1 and PC2) represent 61.2 % of the variance in a and 60.5 % in b. The graph includes primary hepatocytes in 2D monolayer confluent (M _C) or subconfluent (M _S), and 3D (sandwich; S) cultures. The largest deviations from the reference state freshly isolated hepatocytes (FH) occur during the first 24 h of culture in both mouse and human hepatocytes. Moreover, liver tissue (liver) was included into the expression profiling study. A cultivation period from day 1 to day 14 was analyzed for humans. Analysis of the faster dedifferentiating mouse hepatocytes was already terminated at cultivation day 7. The dashed arrows indicate the trend of gene expression changes in time, in relation to freshly isolated primary hepatocytes. c Deregulated genes in a day-by-day analysis of mouse and human hepatocytes. The number of differentially expressed genes (DEGs) is maximally induced at 24 h in culture (‘Day 1 vs FH’). d Cell identity analysis based on gene regulatory network scores (GRN) in freshly isolated and cultivated primary mouse and human hepatocytes. The ‘liver’ GRN score (maximal in fresh hepatocytes) is progressively suppressed during cultivation of mouse hepatocytes in monolayer culture. Sandwich cultures in mouse and human hepatocytes more efficiently sustain the ‘liver’ score. The opposite trend is observed for the ‘fibroblast’ GRN score that increases during cultivation in monolayers, while sandwich cultures ameliorate this effect, whereby that stabilizing effect of collagen sandwiches is strongest in human hepatocytes. Dark blue bars represent the score of the training datasets for ‘liver’ and ‘fibroblast,’ respectively, and the numbers below the bars indicate the days in culture

Gene network activity in cultivated primary hepatocytes is highly similar to diseased mammalian liver tissue

Arch Toxicol. 2016;90(10):2513-2529.

Fig. 2

Comparison of primary hepatocytes in culture to hepatocyte cell lines and stem cell-derived hepatocyte-like cells. a Principal component analysis representing the 1000 genes with highest variance in cultivated mouse hepatocytes, the AML12 cell line at 3 and 24 h in culture and human-induced pluripotent stem cell-derived hepatocyte-like cells (iHep). For mouse, the time course expression alterations are indicated by dashed arrows. The graph shows the top two principal components (PC1 and PC2) representing 66.2 % of the variance. b Principal component analysis representing the 1000 genes with highest variance in cultivated human hepatocytes, the hepatoma cell lines HepaRG and HepG2 at 12 and 24 h in culture, and embryonic stem cell (ESC) as well as human-induced pluripotent stem cell (hiPSC)-derived hepatocyte-like cells (HLC). The graph shows the top two principal components (PC1 and PC2) representing 78.4 % of the variance. c, d Cell/tissue identity analysis based on gene regulatory networks (GRN) in freshly isolated and cultivated primary mouse (c) and human (d) hepatocytes, their corresponding cell lines and hepatocyte-like cells. For mouse and human cell lines, the ‘liver’ GRN scores are significantly lower than in long-term cultured primary hepatocytes. Similarly, the ‘fibroblast’ GRN scores are higher in all cell lines as in long-term cultured hepatocytes. e, f Cell/tissue identity analysis by CellNet in cultivated mouse (e) and human (f) primary hepatocytes, hepatocyte cell lines and stem cell-derived hepatocyte-like cells. The heat maps show the cell and tissue classification probability on freshly isolated primary hepatocytes (FH) and primary hepatocytes in monolayer confluent, subconfluent and sandwich culture for the indicated time (days). Cell/tissue identities based on gene expression profiles were analyzed with the CellNet algorithm (see supplemental methods) and compared to the training expression profiles defining 16 (human) or 20 (mouse) tissues or cells, as described in Cahan et al. ()

Gene network activity in cultivated primary hepatocytes is highly similar to diseased mammalian liver tissue

Arch Toxicol. 2016;90(10):2513-2529.

Fig. 8

Interventions to reduce cultivation-dependent transcriptional alterations in primary hepatocytes. a FACS analysis of cell suspensions from perfused mouse liver before and after three purifications with Percoll gradients. The scatter plots indicate size (FSC-H) and granularity (SSC-H). The populations representing hepatocytes and non-parenchymal cells (NPCs) plus debris are marked, and their corresponding ratios are shown in blue for hepatocytes and red for NPCs. The fraction of NPCs is drastically reduced after successive purifications with Percoll. b Real-time qPCR analysis of the diagnostic genes for ‘inflammation’ (Lcn2, Saa3) and ‘mature liver functions’ (Bsep, Mrp2) in hepatocytes on monolayer culture (day 1), without or after 1, 2 or 3 purifications with Percoll gradient centrifugation. c Confocal images of mouse hepatocytes stained with phalloidin (red), anti-DPPIV antibodies (green) and DAPI (blue), showing polymerized actin, apical domains and nuclei, respectively. The analysis was performed on hepatocytes in monolayer confluent (M _C), sandwich (S) and spheroid cultures. The spheroid was stained at day 5 in culture. d Real-time qPCR analysis of the diagnostic genes for the ‘inflammation’ (Lcn2) and ‘mature liver functions’ (Bsep) in hepatocytes cultivated on different matrix supports. The top graphs show the expression levels of diagnostic genes in sandwich (S), monolayer confluent (M _C) and spheroids (Sph) at days 1 and 5. The bottom graphs show expression levels of diagnostic genes in hepatocytes cultured on collagen coated monolayer confluent (monolayer), collagen sandwich (sandwich), matrigel and laminin at the indicated cultivation periods. e Real-time qPCR analysis of the diagnostic genes for ‘inflammation’ (Lcn2) and ‘mature liver functions’ (Bsep), in hepatocytes cultures on collagen monolayer using standard culture media (control) or additional bile salts for 24 h. DMSO was included as vehicle control. UCDA ursodeoxycholic acid, CA cholic acid, DCA deoxycholic acid, TCA-Na taurocholic acid, TLCA taurolithocholic acid, GDCA glycodeoxycholic acid, CDCA chenodeoxycholic acid (*p < 0.05 compared to controls, Mann–Whitney test, two-sided)

Gene network activity in cultivated primary hepatocytes is highly similar to diseased mammalian liver tissue

Arch Toxicol. 2016;90(10):2513-2529.