Generation and characterization of mature hepatocyte organoids for liver metabolic studies
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE245632 | GEO | 2024/05/15
REPOSITORIES: GEO
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Project description:Human hepatocytes differ in gene expression and function across the hexagonal lobules of the tissue microarchitecture, a phenomenon referred to as liver zonation. Hepatocytes also display intra-lobular differences in cell size, but to what extent zonal expression and function correlate with cell size in isolated human hepatocytes is not entirely clear. Here, we first used our accumulated experience of nearly 100 hepatocyte isolations to assess the impact of donor background and process parameters on hepatocyte quality. We observed substantial inter-batch variability in cell size distributions and a tendency for overall cell size to affect the outcome of hepatocyte isolation and cryopreservation. We further separated cells into different size fractions and analyzed them with label-free quantitative proteomics. This showed that protein abundances in different hepatocyte size fractions recapitulated the in vivo expression patterns of well-known zonal markers. We also found that proteins with sequential enrichment across fractions largely represented biological processes with known zonal specificity. This was confirmed by differences in the metabolic activity of zonated CYP enzymes. Altogether, our results show that hepatocyte size corresponds to zonal origin, and that our size fractionation approach can be used to study zone-specific liver functions in vitro.
2021-01-26 | PXD013142 | Pride
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.
2024-08-22 | GSE182603 | 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:Human liver organoids are expected to be a hepatocyte source for preclinical in vitro studies. Although these organoids show long-term proliferation, their hepatic functions remain low. Here, we propose a novel method for two dimensional (2D)-cultured hepatic differentiation from human liver organoids. When cultured under a 2D condition, the single cells from human liver organoids were seeded on collagen type I-coated plates. Then, optimal conditions for hepatic differentiation were screened using several reagents. We determined the 2D-cultured hepatocyte differentiation method from human liver organoids. Hepatic gene expressions in human liver organoids-derived hepatocytes (Org-HEPs) were greatly increased, compared to those in human liver organoids. The metabolic activities of cytochrome P450 (CYP) 1A2, CYP2C8, CYP2E1 and CYP3A4 were at levels comparable to those in primary human hepatocytes (PHHs). These results suggested that human liver organoids could be differentiated into highly functional hepatocytes in 2D culture. We also treated Org-HEPs and PHHs with hepatotoxic drugs. The cell viability of Org-HEPs was almost the same as that of PHHs, suggesting that Org-HEPs could be used for hepatotoxicity tests. Thus, Org-HEPs will be useful for pharmaceutical research.
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Project description:Human liver organoids, an in vitro 3D culture system to recapitulate biological tissue, are expected to be used for drug discovery. However, Matrigel, the most widely used extracellular matrix for organoid culture, has concerns about safety and reproducibility since it is murine-derived. Morever, low hepatic functions of human liver organoids compared to primary human hepatocytes is considered a challenge. Herein, we attempted to culture human liver organoids, established from primary (cryopreserved) human hepatocytes (PHH), using HYDROX, a chemically defined 3D nanofiber. While proliferative capacity of human liver organoids was lost by HYDROX-culture, the gene expression level of a hepatocyte marker CYP3A4 and the CYP3A4 metabolic activity in HYDROX-cultured liver organoids were significantly improved, comparable to those of PHH. HYDROX-cultured liver organoids when treated with hepatotoxic drugs such as acetaminophen showed similar cell viability to that of PHH, suggesting that HYDROX-cultured liver organoids could be applied to drug-induced hepatotoxicity test. Furthermore, HYDROX-cultured liver organoids maintained its functions for up to 35 days and could be used to estimate chronic drug-induced hepatotoxicity such as those of fialuridine. Our findings demonstrated that human liver organoids obtained high liver functions by HYDROX-culture, meaning that HYDROX could contribute to drug discovery as a novel biomaterial.
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Project description:Hepatic steatosis is the result of an imbalance between nutrient delivery and metabolism in the liver. It is the first hallmark of Non-alcoholic fatty liver disease (NAFLD) and is characterized by the accumulation of excess lipids in the liver that can drive liver failure, inflammation, and cancer. Mitochondria control the fate and function of cells and compelling evidence implicates these multifunctional organelles in the appearance and progression of liver dysfunction, although it remains to be elucidated which specific mitochondrial functions are actually causally linked to NAFLD. Here, we identified Mitochondrial Fission Process 1 protein (MTFP1) as a key regulator of mitochondrial and metabolic activity in the liver. Deletion of Mtfp1 in hepatocytes is physiologically benign in mice yet leads to the upregulation of oxidative phosphorylation (OXPHOS) complexes and mitochondrial respiration, independently of mitochondrial biogenesis. Consequently, hepatocyte-specific knockout mice are protected against high fat diet-induced hepatic steatosis and metabolic dysregulation. Additionally, we find that deletion of Mtfp1 in liver mitochondria inhibits mitochondrial permeability transition pore opening in hepatocytes, conferring protection against apoptotic liver damage in vivo and ex vivo. Our work uncovers novel functions of MTFP1 in the liver, positioning this gene as an unexpected regulator of OXPHOS and a therapeutic candidate for NAFLD.
2023-11-27 | PXD046262 | Pride
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