Exploring the impact of alcohol exposure on hiPSC-derived hepatocytes with PPA2 mutation : insights from immunofluorescence and gene expression analysis
Kovalevskaya, Sofya (2025)
2025
Master's Programme in Biomedical Sciences and Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2025-11-27
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-2025112610926
https://urn.fi/URN:NBN:fi:tuni-2025112610926
Tiivistelmä
Mitochondrial dysfunction plays a key role in many metabolic and degenerative diseases, including those caused by alcohol consumption. The PPA2 gene encodes a mitochondrial inorganic pyrophosphatase critical for ATP homeostasis and energy metabolism. Mutations in PPA2 have been primarily associated with sudden cardiac death, but their role in hepatic metabolism and alcohol response has not been intensively researched. The aim of this study was to investigate how ethanol (EtOH) exposure affects the viability, mitochondrial function, and differentiation behaviour of PPA2-mutated and wild-type human induced pluripotent stem cell (hiPSC)-derived hepatocyte-like cells (HLCs).
PPA2-mutated and wild-type cell lines were differentiated into HLCs following a three-stage hepatic induction protocol. The differentiation efficiency was verified by characteristic morphology and expression of hepatic markers. Cells were subsequently exposed to two EtOH concentrations (0,5‰ and 3,0‰) for 48 and 120 hours, representing mild to moderate alcohol exposure. Viability was assessed by fluorescent live/dead staining, while mitochondrial functionality was evaluated using MitoTracker. Immunofluorescence staining and qPCR were performed to quantify changes in hepatic and mitochondrial gene and protein expression profiles.
The results demonstrated that EtOH exposure up to 3,0‰ didn’t induce substantial cytotoxicity or mitochondrial depolarization in either line. Both wild-type and PPA2-mutant cells retained hepatic morphology and stable mitochondrial function, indicating resilience against moderate EtOH stress. Gene expression analysis revealed moderate transcriptional alterations, including variable regulation of AFP, ALB, CYP2E1, and PPA2, suggesting activation of adaptive metabolic mechanisms rather than direct cytotoxic effects. Interestingly, spontaneous cell contractility was observed in the PPA2-mutant line, independent of EtOH treatment, implying a potential lineage deviation toward cardiomyocyte-like cells and highlighting the importance of PPA2 in mitochondrial and energetic regulation during differentiation.
These findings suggest that PPA2 mutation alone doesn’t make HLCs more vulnerable to alcohol-induced mitochondrial dysfunction or death, but it may still alter their differentiation path by causing energetic and transcriptional shifts. The study also emphasizes the methodological challenges of maintaining stable EtOH exposure in vitro and underscores the need for improved experimental models, such as microfluidic liver-on-chip systems.
Future research should focus on using three dimensional (3D) hepatic models to improve EtOH diffusion control, evaluating higher EtOH concentrations, and performing multiomics analyses to uncover PPA2-related signaling mechanisms. Further investigation into the spontaneous contractility phenotype is also needed to determine whether it represents a stable cardiomyocyte-like cell lineage commitment.
PPA2-mutated and wild-type cell lines were differentiated into HLCs following a three-stage hepatic induction protocol. The differentiation efficiency was verified by characteristic morphology and expression of hepatic markers. Cells were subsequently exposed to two EtOH concentrations (0,5‰ and 3,0‰) for 48 and 120 hours, representing mild to moderate alcohol exposure. Viability was assessed by fluorescent live/dead staining, while mitochondrial functionality was evaluated using MitoTracker. Immunofluorescence staining and qPCR were performed to quantify changes in hepatic and mitochondrial gene and protein expression profiles.
The results demonstrated that EtOH exposure up to 3,0‰ didn’t induce substantial cytotoxicity or mitochondrial depolarization in either line. Both wild-type and PPA2-mutant cells retained hepatic morphology and stable mitochondrial function, indicating resilience against moderate EtOH stress. Gene expression analysis revealed moderate transcriptional alterations, including variable regulation of AFP, ALB, CYP2E1, and PPA2, suggesting activation of adaptive metabolic mechanisms rather than direct cytotoxic effects. Interestingly, spontaneous cell contractility was observed in the PPA2-mutant line, independent of EtOH treatment, implying a potential lineage deviation toward cardiomyocyte-like cells and highlighting the importance of PPA2 in mitochondrial and energetic regulation during differentiation.
These findings suggest that PPA2 mutation alone doesn’t make HLCs more vulnerable to alcohol-induced mitochondrial dysfunction or death, but it may still alter their differentiation path by causing energetic and transcriptional shifts. The study also emphasizes the methodological challenges of maintaining stable EtOH exposure in vitro and underscores the need for improved experimental models, such as microfluidic liver-on-chip systems.
Future research should focus on using three dimensional (3D) hepatic models to improve EtOH diffusion control, evaluating higher EtOH concentrations, and performing multiomics analyses to uncover PPA2-related signaling mechanisms. Further investigation into the spontaneous contractility phenotype is also needed to determine whether it represents a stable cardiomyocyte-like cell lineage commitment.