In the classical form of α1‐antitrypsin deficiency (ATD), aberrant intracellular accumulation of misfolded mutant α1‐antitrypsin Z (ATZ) in hepatocytes causes hepatic damage by a gain‐of‐function, “proteotoxic” mechanism. Whereas some ATD patients develop severe liver disease (SLD) that necessitates liver transplantation, others with the same genetic defect completely escape this clinical phenotype. We investigated whether induced pluripotent stem cells (iPSCs) from ATD individuals with or without SLD could model these personalized variations in hepatic disease phenotypes. Patient‐specific iPSCs were generated from ATD patients and a control and differentiated into hepatocyte‐like cells (iHeps) having many characteristics of hepatocytes. Pulse‐chase and endoglycosidase H analysis demonstrate that the iHeps recapitulate the abnormal accumulation and processing of the ATZ molecule, compared to the wild‐type AT molecule. Measurements of the fate of intracellular ATZ show a marked delay in the rate of ATZ degradation in iHeps from SLD patients, compared to those from no liver disease patients. Transmission electron microscopy showed dilated rough endoplasmic reticulum in iHeps from all individuals with ATD, not in controls, but globular inclusions that are partially covered with ribosomes were observed only in iHeps from individuals with SLD. Conclusion: iHeps model the individual disease phenotypes of ATD patients with more rapid degradation of misfolded ATZ and lack of globular inclusions in cells from patients who have escaped liver disease. The results support the concept that “proteostasis” mechanisms, such as intracellular degradation pathways, play a role in observed variations in clinical phenotype and show that iPSCs can potentially be used to facilitate predictions of disease susceptibility for more precise and timely application of therapeutic strategies. (Hepatology 2015;62:147‐157)

Hepatocyte transplantation has the potential to cure inherited liver diseases, but its application is impeded by a scarcity of donor livers. Therefore, we explored whether transplantation of hepatocyte-like cells (iHeps) differentiated from human induced pluripotent stem cells (iPSCs) could ameliorate inherited liver diseases. iPSCs reprogrammed from human skin fibroblasts were differentiated to iHeps, which were transplanted into livers of uridinediphosphoglucuronate glucuronosyltransferase-1 (UGT1A1)-deficient Gunn rats, a model of Crigler-Najjar syndrome 1 (CN1), where elevated unconjugated bilirubin causes brain injury and death. To promote iHep proliferation, 30% of the recipient liver was X-irradiated before transplantation, and hepatocyte growth factor was expressed. After transplantation, UGT1A1+ iHep clusters constituted 2.5%-7.5% of the preconditioned liver lobe. A decline of serum bilirubin by 30%-60% and biliary excretion of bilirubin glucuronides indicated that transplanted iHeps expressed UGT1A1 activity, a postnatal function of hepatocytes. Therefore, iHeps warrant further exploration as a renewable source of hepatocytes for treating inherited liver diseases.

Hepatocyte transplantation has been used to treat liver disease. The availability of cells for these procedures is quite limited. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be a useful source of hepatocytes for basic research and transplantation if efficient and effective differentiation protocols were developed and problems with tumorigenicity could be overcome. Recent evidence suggests that the cell of origin may affect hiPSC differentiation. Thus, hiPSCs generated from hepatocytes may differentiate back to hepatocytes more efficiently than hiPSCs from other cell types. We examined the efficiency of reprogramming adult and fetal human hepatocytes. The present studies report the generation of 40 hiPSC lines from primary human hepatocytes under feeder-free conditions. Of these, 37 hiPSC lines were generated from fetal hepatocytes, 2 hiPSC lines from normal hepatocytes, and 1 hiPSC line from hepatocytes of a patient with Crigler–Najjar syndrome, type 1. All lines were confirmed reprogrammed and expressed markers of pluripotency by gene expression, flow cytometry, immunocytochemistry, and teratoma formation. Fetal hepatocytes were reprogrammed at a frequency over 50-fold higher than adult hepatocytes. Adult hepatocytes were only reprogrammed with six factors, while fetal hepatocytes could be reprogrammed with three (OCT4, SOX2, NANOG) or four factors (OCT4, SOX2, NANOG, LIN28 or OCT4, SOX2, KLF4, C-MYC). The increased reprogramming efficiency of fetal cells was not due to increased transduction efficiency or vector toxicity. These studies confirm that hiPSCs can be generated from adult and fetal hepatocytes including those with genetic diseases. Fetal hepatocytes reprogram much more efficiently than adult hepatocytes, although both could serve as useful sources of hiPSC-derived hepatocytes for basic research or transplantation.

The only definitive therapy for end‐stage liver disease is whole‐organ transplantation. The success of this intervention is severely limited by the complexity of the surgery, the cost of patient care, the need for long‐term immunosuppression, and the shortage of donor organs. In rodents and humans, end‐stage degeneration of hepatocyte function is associated with disruption of the liver‐specific transcriptional network and a nearly complete loss of promoter P1‐driven hepatocyte nuclear factor 4‐alpha (P1‐HNF4α) activity. Re‐expression of HNF4α2, the predominant P1‐HNF4α, reinstates the transcriptional network, normalizes the genes important for hepatocyte function, and reverses liver failure in rodents. In this study, we tested the effectiveness of supplementary expression of human HNF4α2 messenger RNA (mRNA) in primary human hepatocytes isolated from explanted livers of patients who underwent transplant for end‐stage irreversibly decompensated liver failure (Child‐Pugh B, C) resulting from alcohol‐mediated cirrhosis and nonalcoholic steatohepatitis. Re‐expression of HNF4α2 in decompensated cirrhotic human hepatocytes corrects the disrupted transcriptional network and normalizes the expression of genes important for hepatocyte function, improving liver‐specific protein expression. End‐stage liver disease in humans is associated with both loss of P1‐HNF4α expression and failure of its localization to the nucleus. We found that while HNF4α2 re‐expression increased the amount of P1‐HNF4α protein in hepatocytes, it did not alter the ability of hepatocytes to localize P1‐HNF4α to their nuclei. Conclusion: Re‐expression of HNF4α2 mRNA in livers of patients with end‐stage disease may be an effective therapy for terminal liver failure that would circumvent the need for organ transplantation. The efficacy of this strategy may be enhanced by discovering the cause for loss of nuclear P1‐HNF4α localization in end‐stage cirrhosis, a process not found in rodent studies.

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Metabolic-dysfunction-associated steatotic liver disease (MASLD), which affects 30 million people in the US and is anticipated to reach over 100 million by 2030, places a significant financial strain on the healthcare system. There is presently no FDA-approved treatment for MASLD despite its public health significance and financial burden. Understanding the connection between point mutations, liver enzymes, and MASLD is important for comprehending drug toxicity in healthy or diseased individuals. Multiple genetic variations have been linked to MASLD susceptibility through genome-wide association studies (GWAS), either increasing MASLD risk or protecting against it, such as PNPLA3 rs738409, MBOAT7 rs641738, GCKR rs780094, HSD17B13 rs72613567, and MTARC1 rs2642438. As the impact of genetic variants on the levels of drug-metabolizing cytochrome P450 (CYP) enzymes in human hepatocytes has not been thoroughly investigated, this study aims to describe the analysis of metabolic functions for selected phase I and phase II liver enzymes in human hepatocytes. For this purpose, fresh isolated primary hepatocytes were obtained from healthy liver donors (n = 126), and liquid chromatography–mass spectrometry (LC–MS) was performed. For the cohorts, participants were classified into minor homozygotes and nonminor homozygotes (major homozygotes + heterozygotes) for five gene polymorphisms. For phase I liver enzymes, we found a significant difference in the activity of CYP1A2 in human hepatocytes carrying MBOAT7 (p = 0.011) and of CYP2C8 in human hepatocytes carrying PNPLA3 (p = 0.004). It was also observed that the activity of CYP2C9 was significantly lower in human hepatocytes carrying HSD17B13 (p = 0.001) minor homozygous compared to nonminor homozygous. No significant difference in activity of CYP2E1, CYP2C8, CYP2D6, CYP2E1, CYP3A4, ECOD, FMO, MAO, AO, and CES2 and in any of the phase II liver enzymes between human hepatocytes carrying genetic variants for PNPLA3 rs738409, MBOAT7 rs641738, GCKR rs780094, HSD17B13 rs72613567, and MTARC1 rs2642438 were observed. These findings offer a preliminary assessment of the influence of genetic variations on drug-metabolizing cytochrome P450 (CYP) enzymes in healthy human hepatocytes, which may be useful for future drug discovery investigations.