In Vitro Modeling of Alcohol-Induced Liver Injury Using Human-Induced Pluripotent Stem Cells.
- Authors
- Tian, Lipeng; Prasad, Neha; Jang, Yoon-Young
- Year
- 2016
- Journal
- Methods in molecular biology (Clifton, N.J.)
- PMID
- 25520290
- DOI
- 10.1007/7651_2014_168
- PMCID
- PMC5881111
Alcohol consumption has long been associated with a majority of liver diseases and has been found to influence both fetal and adult liver functions. In spite of being one of the major causes of morbidity and mortality in the world, currently, there are no effective strategies that can prevent or treat alcoholic liver disease (ALD), due to a lack of human-relevant research models. Recent success in generation of functionally active mature hepatocyte-like cells from human-induced pluripotent cells (iPSCs) enables us to better understand the effects of alcohol on liver functions. Here, we describe the method and effect of alcohol exposure on multistage hepatic cell types derived from human iPSCs, in an attempt to recapitulate the early stages of liver tissue injury associated with ALD. We exposed different stages of iPSC-induced hepatic cells to ethanol at a pathophysiological concentration. In addition to stage-specific molecular markers, we measured several key cellular parameters of hepatocyte injury, including apoptosis, proliferation, and lipid accumulation.
Differentiation of human iPSCs into multistage hepatic cells. (a) A schematic diagram of hepatic differentiation procedure and corresponding bright field images of hepatic cells at each stage. (b) A representative image showing the expression of Sox17 (red), a definitive endoderm marker, at differentiation day 4. (c) Flow cytometric analysis shows that ~99 % of cells express CXCR4, another definitive endoderm marker, at day 4. (d) Expression of AFP (green), a hepatocyte progenitor marker, at day 9 after initiation of hepatic differentiation. (e and f) At day 20, most of the cells express mature hepatocyte markers such as albumin (ALB, green) and alpha-1 antitrypsin (AAT, red). Scale bars, 100 μm
Effects of alcohol on hepatic differentiation and cell viability. (a) Real-time PCR analysis of Sox17 expression (left panel) at day 4 definitive endoderm (DE) stage, following 100 mM ethanol treatment from day 0 to day 3. Percentage of Annexin V-positive cells was obtained from analysis of cell apoptosis by flow cytometry (right panel). Apoptotic cells were increased with alcohol exposure at DE stage. (b) Significant reduction in AFP expression (left panel) was observed in hepatic progenitor (HP) cells at day 9, on exposure to 100 mM ethanol from day 4 to day 8, and the quantity of Annexin V-positive apoptotic cells also increased significantly (right panel). (c) Alcohol treatment from day 11 to day 15, followed by analysis of albumin (ALB) expression (left panel) and cell viability (right panel) of the mature hepatocyte-like cells at day 16, showed no statistical difference compared with control. *: p < 0.05, #: p < 0.01
Effects of alcohol on proliferation of iPSC-derived multistage hepatic cells. (a) Representative images of Ki67 (Red)-positive cells at day 4 DE stage, (b) Ki67 (green)-positive cells at day 9 HP stage, (c) ALB-positive cells (green), and Ki67 (red)-positive cells at day 16 MH stage in control and ethanol-treated cells. Scale bars, 100 μm
Ethanol exposure induces fat accumulation in differentiated hepatocytes from iPSCs. (a and b) Detection of lipid droplets by Oil Red O. (c) Quantification of lipid accumulation with Oil Red O staining. (d) Real-time PCR analysis of expression levels of fatty acid synthase (FASN) in control and 100 mM ethanol-treated groups. *: p < 0.05. Scale bars, 100 μm
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| From Cells to Organs: The Present and Future of Regenerative Medicine. | Wang Y et al. | — | 2022 | → |
| Biochemical Mechanisms Associating Alcohol Use Disorders with Cancers. | Rodriguez FD et al. | — | 2021 | → |
| Bioengineered Liver Models for Investigating Disease Pathogenesis and Regenerative Medicine. | Kukla DA et al. | — | 2021 | → |
| A comparison of hepato-cellular in vitro platforms to study CYP3A4 induction. | Bulutoglu B et al. | — | 2020 | → |
| <i>In vitro</i>nonalcoholic fatty liver disease model with cyclo-olefin-polymer-based microphysiological systems | Wen X et al. | — | 2020 | — |
| Human-relevant preclinical in vitro models for studying hepatobiliary development and liver diseases using induced pluripotent stem cells. | Chaudhari P et al. | — | 2019 | → |
| Stem cells under the influence of alcohol: effects of ethanol consumption on stem/progenitor cells. | Di Rocco G et al. | — | 2019 | → |
| Using human stem cells as a model system to understand the neural mechanisms of alcohol use disorders: Current status and outlook. | Scarnati MS et al. | — | 2019 | → |
| Induced Pluripotent Stem Cell-Derived Hepatocytes and Precision Medicine in Human Liver Disease. | Kulkarni S et al. | — | 2018 | → |
| Alcohol Increases Liver Progenitor Populations and Induces Disease Phenotypes in Human IPSC-Derived Mature Stage Hepatic Cells. | Tian L et al. | — | 2016 | → |
| Current Management of Alcoholic Hepatitis and Future Therapies. | Saberi B et al. | — | 2016 | → |
| Efficient and Controlled Generation of 2D and 3D Bile Duct Tissue from Human Pluripotent Stem Cell-Derived Spheroids. | Tian L et al. | — | 2016 | → |
| Stem cell-derived liver cells for drug testing and disease modeling. | Davidson MD et al. | — | 2015 | → |