Unknown,Transcriptomics,Genomics,Proteomics

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Comprehensive expression analysis of primary hepatocytes under four different culture conditions

ABSTRACT: Primary hepatocytes have been widely explored as cell sources for the study of in vitro drug metabolism and pharmacokinetics (DMPK). Aiming toward establishing an in vitro drug screening method, the current study illustrated a comprehensive increase in the DMPK-related gene expression of nanopillar (NP)-cultured 3D-spheroid. To examine the expressional changes in DMPK-related genes under four different conditions, namely, NP-, sandwich (SW)-, monolayer (ML)-cultured rat hepatocytes, and freshly isolated hepatocytes, genome-wide gene-expression analysis using a DNA microarray was performed. Among the DMPK-related genes, cytochrome P450, UDP-glucuronosyltransferase, and transporter genes were focused on. Principal component analysis showed that the global gene expression profile in sample from NP culture is closer to that from freshly isolated hepatocytes than that from SW culture. The expressions of almost all Cyp 1 to 3 and Ugt genes of NP-cultured 3-D spheroid were higher than those of ML and SW. The expression of Abcc2 gene whose translation product has a critical role in excretion of metabolized bile acids in hepatocyte to bile canaliculi was three times higher in NP than in ML. From these results, 3-D spheroid formed by the NP culture was suggested to possess higher ability of metabolism and excretion than conventional 2-D monolayer culture. The NP culture has a potential as an alternative culturing technique for evaluating metabolism and toxicity toward the development of new drugs. Gene expression in rat hepatocyte was measured under four different conditions, namely, Nanopillar (NP)-, sandwich (SW)-, monolayer (ML)-cultured rat hepatocytes, and freshly isolated hepatocytes. Three independent experiments were performed at 95 hours of post-seeding.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Ryosuke Takahashi

PROVIDER: E-GEOD-38950 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The liver is one of most important organs in our bodies. It performs many essential functions including metabolism, synthesis, secretion, detoxification, and storage. Hepatocytes are the principal cell type in the liver and are involved in multiple liver-specific functions. There have been several efforts to develop in vitro culture systems capable of maintaining hepatocyte-specific phenotype over long time periods. In hepatic tissue engineering, two commonly used culture systems are the collagen sandwich and monolayers of cells. ‚àö√á¬¨‚Ä†In this study, genome-wide gene expression profiles of primary hepatocytes were measured over an 8-day period for each cell culture system using Affymetrix GeneChips and analyzed via Gene Set Enrichment Analysis (GSEA), which is a powerful method to elicit biologically meaningful information from microarray data at the level of gene sets. Results indicate that the gene expression in hepatocytes in collagen sandwich cultures gradually diverges from that in monolayer cultures. Gene sets up-regulated in collagen sandwich cultures include those associated with liver metabolic and synthetic functions. ‚àö√á¬¨‚Ä†These functions are associated with lipid, amino acid, carbohydrate, and alcohol metabolism and bile acid synthesis. Nuclear receptors are up-regulated in collagen sandwiches 24 hours after seeding. Signals transmitted from these receptors may cause the up-regulation of other processes in subsequent days. Cytochrome-P450 monooxygenase expression was initially down-regulated but exhibited up-regulation after 72 hours. Our results provide a baseline for further explorations into the systems biology of engineered liver mimics as well as 2D and 3D co-cultures of primary hepatocytes and non-parenchymal cells. To better understand differences in quality of in vitro growth of rat hepatocytes between culture on a monolayer of collagen gel and sandwiched between two layers of gel, we measured gene expression in hepatocytes in these two culture conditions in triplicate for four time points: 1 day, 2 days, 3 days, and 8 days of culturing. Overall, we obtained Affymetrix microarray data for 24 samples, divided to 12 samples from monolayer and double layer cultures each, each of which are divided into four time points with 3 sample replicates.

Project description:Compared to conventional monolayer cell culture, three-dimensional (or spheroid) cultures are more reflective of the in vivo environment and represent a better means of representing normal tissue. Therefore, understanding the biology of spheroid-derived cells is important for a more complete appreciation of in vivo tissue function. Although it has been shown that culture conditions such as nutrients, oxygen, cell passage etc. alter gene expression patterns of the cells, the gene expression profile of spheroid as compared to conventional monolayer culture in primary cells has not been studied. To investigate the effects of spheroid culture system on gene expression of primary cells, single cell RNA sequencing (scRNAseq) was performed on mouse dermal fibroblasts (tail/ear fibroblasts, TEFs) cultured as monolayers on cell culture dishes or grown as spheroids. After quality control, a total of 11,515 cells (8,022 spheroid cells and 3,493 cells of monolayer fibroblasts) were analyzed using Uniform Manifold Approximation and Projection (UMAP) which identified 2 largely separated libraries. These libraries contained cells with 2,491 and 8722 detected genes (nFeature_RNA) for monolayer and spheroid cells respectively. UMAP clustering of the integrated datasets identified 8 significant cell clusters (resolution=0.3) in which clusters 0, 1, 2, 4 and 7 had a higher number of cells in the spheroid library. Differential gene expression analyses revealed that each cluster was characterized by a specific transcriptional profile. Among cell clusters, cluster 4 exhibited significantly increased expression of collagen family genes, including Col1a2, Col3a1, Col1a1, Col5a2, Col4a, Col4a2, Col5a3, Col5a1, Col6a1, Col12a1 and Col15a1. Furthermore, expression of mesenchymal stem cell genes [Sca-1 (Ly6a), CD29 (Itgb1), CD44, CD90.1 (Thy1)] as well as a group of genes important for stem cell self-renewal including Notch2, Sox4, Sox9 Klf2, and Foxp1 were significantly increased in cluster 4. Immunofluorescence (IF) staining was performed to validate the protein expression of selected genes. Our work provides a significant advance in characterizing the global gene expression profile of spheroid culture of mouse TEFs compared to monolayer culture. We reached a high resolution at single-cell level, which enabled us to identify specific clusters associated with mesenchymal stem-like cells.

Project description:It is well-known that isolation and cultivation of primary hepatocytes causes 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 one day cultivation and one 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 minority 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, (3) upregulated migration/cell-cycle associated genes. Gene regulatory network analysis highlights overrepresented and deregulated HNF4 and CAR (cluster 1), KrÃ¼ppel-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 by far not abrogating cultivation-induced responses include removal of non-parenchymal cells, generation of the hepatocytesâ own matrix in spheroids, supplementation with bile salts and siRNA mediated suppression of key transcription factors. In conclusion, the 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.

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Comprehensive expression analysis of primary hepatocytes under four different culture conditions

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