Integrative proteomics of primary hepatocytes during dedifferentiation
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PROVIDER: PRJNA574425 | ENA |
REPOSITORIES: ENA
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Project description:To understand the relationship between protein expression and mRNA translation during primary hepatocytes dedifferentiation, we have employed transcriptome microarrayas a discovery platform. Rat primary hepatocytes were isolated by the method of two-step enzymes perfusion and then cultured on mono-layer in vitro. Samples at 0h( just after perfusion, before planking) , 6h, 12h ,24h and 48h were collected. Integrative analysis of transcriptome and whole cell proteomics (WCP) leaded us to realize the poor correlation of them. This discovery made us realize that targeting mRNA was far from enough in illustrating this process. It would provide new insights from the aspects of post-translational modifications(PTMs).Post-translational modifications play important role in numorous biological and pathological process, but a few reports are related to primary hepatocytes dedifferentiation process, and there is still no integrative proteomics analysis in this field yet. In this study, we perform ubiquitinome phosphorylated proteome, whole cell proteome and transcriptome simultaneously during the five different time points of dedifferentiation in vitro quantified over 6000 modified sites mapping to over 2000 proteins. And comprehensive analysis of these datasets provides novel insight in this field.
2022-09-26 | GSE138071 | GEO
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Project description:Primary hepatocytes are a commonly used in vitro model for studying liver metabolism, but prolonged culturing results in dedifferentiation. Here we characterized the transcriptome and proteome of whole liver and primary hepatocytes as either freshly isolated cells or after 24 hours of 2D-culturing. We found that after 24 hours of 2D-culturing, hepatocytes showed differential expression of over 10,000 genes and 3,000 proteins compared to freshly isolated cells. This was accompanied by a reduced transcriptional heterogeneity and a loss of zonal markers. Cultures contained ~10% non-hepatocytes, including stellate- and dedifferentiated endothelial cells. Culturing also increased abundance of proteins associated with oxidative stress and inflammation, and changed mitochondrial, extracellular, and ribosomal protein abundance. The data generated are available in the shiny-app “Hepamorphosis, https://cbmr-rmpp.shinyapps.io/hepamorphosis/”. Our finding show that primary mouse hepatocytes undergo significant changes in culture, limiting their utility for studying physiological and molecular mechanisms related to the liver.
2025-07-07 | GSE280301 | GEO
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Project description:Advances in 3D cell culture systems, including the hepatic organoids, has shown the potential to model the liver development in vitro. However, hepatocyte-like cells obtained by these means are still immature compared to the primary human hepatocytes. Here we applied single-cell RNA-seq and ATAC-seq to identify gene regulatory mechanisms distinct to mature human hepatocytes (in vivo) and organoid derived hepatocyte like cells (in vitro). Using a modified two-step perfusion protocol, primary hepatocytes from all lobular zones were obtained with high purity. Integrative analysis revealed a key role of transcription factors of the AP-1 family in cooperation with hepatocyte-specific transcription factors, such as HNF4A, in establishing cellular identity of mature hepatocytes. Furthermore, it revealed distinct transcription factor sets required/active in human hepatocytes and liver organoids. Function analysis of an organoid-enriched transcription factor ELF3, showed that decreased expression of ELF3 in liver organoids promotes increased expression of genes typical to mature hepatocytes. This study indicates that ELF3 functions as a barrier of hepatocyte differentiation from liver organoids. Collectively, our integrative analysis provides critical insights into the transcriptional regulatory networks of human hepatocytes and liver organoids, which will further efficient differentiation of functional hepatocytes in vitro.
2024-08-22 | GSE182604 | GEO
Project description:Advances in 3D cell culture systems, including the hepatic organoids, has shown the potential to model the liver development in vitro. However, hepatocyte-like cells obtained by these means are still immature compared to the primary human hepatocytes. Here we applied single-cell RNA-seq and ATAC-seq to identify gene regulatory mechanisms distinct to mature human hepatocytes (in vivo) and organoid derived hepatocyte like cells (in vitro). Using a modified two-step perfusion protocol, primary hepatocytes from all lobular zones were obtained with high purity. Integrative analysis revealed a key role of transcription factors of the AP-1 family in cooperation with hepatocyte-specific transcription factors, such as HNF4A, in establishing cellular identity of mature hepatocytes. Furthermore, it revealed distinct transcription factor sets required/active in human hepatocytes and liver organoids. Function analysis of an organoid-enriched transcription factor ELF3, showed that decreased expression of ELF3 in liver organoids promotes increased expression of genes typical to mature hepatocytes. This study indicates that ELF3 functions as a barrier of hepatocyte differentiation from liver organoids. Collectively, our integrative analysis provides critical insights into the transcriptional regulatory networks of human hepatocytes and liver organoids, which will further efficient differentiation of functional hepatocytes in vitro.
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Project description:Advances in 3D cell culture systems, including the hepatic organoids, has shown the potential to model the liver development in vitro. However, hepatocyte-like cells obtained by these means are still immature compared to the primary human hepatocytes. Here we applied single-cell RNA-seq and ATAC-seq to identify gene regulatory mechanisms distinct to mature human hepatocytes (in vivo) and organoid derived hepatocyte like cells (in vitro). Using a modified two-step perfusion protocol, primary hepatocytes from all lobular zones were obtained with high purity. Integrative analysis revealed a key role of transcription factors of the AP-1 family in cooperation with hepatocyte-specific transcription factors, such as HNF4A, in establishing cellular identity of mature hepatocytes. Furthermore, it revealed distinct transcription factor sets required/active in human hepatocytes and liver organoids. Function analysis of an organoid-enriched transcription factor ELF3, showed that decreased expression of ELF3 in liver organoids promotes increased expression of genes typical to mature hepatocytes. This study indicates that ELF3 functions as a barrier of hepatocyte differentiation from liver organoids. Collectively, our integrative analysis provides critical insights into the transcriptional regulatory networks of human hepatocytes and liver organoids, which will further efficient differentiation of functional hepatocytes in vitro.
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