Acute liver failure & hepatocyte transplantation
Date Issued
2016
Date
2016
Author(s)
Ho, Cheng-Maw
Abstract
Background and objective Acute liver failure (ALF) is uncommon but fatal. Current management is based mostly on clinical experience. Hepatocyte transplantation is a promising alternative to liver transplantation in patients with acute liver failure. The study is to investigate ALF from longitudinal population-scale epidemiological analysis, through individual cross-sectional histopathophysiological observation, ex vivo functional evaluation of hepatocytes, to preclinical animal experiment of hepatocyte transplantation. To this end, we investigated 1) the incidence, etiology, outcomes, and prognostic factors of ALF in Taiwan. 2) pathophysiological expression of regeneration and differentiation in acute failure liver. 3) whether the uptake and release of indocyanine green (ICG) by hepatocytes could be used as a rapid in vitro assay for hepatocyte functional assessment. 4) the impact of the rate of intraportal hepatocyte transplantation on early engraftment and repopulation and to improve the engraftment and repopulation efficiencies of hepatocyte transplantation for treatment of a rat model of acute liver failure in a clinically useful way without preconditioning. Materials and methods 1) For population study, patients with the admission diagnosis of ALF between January 2005 and September 2007 were identified from the Longitudinal Health Insurance Database of Taiwan. ALF was further confirmed by disease severity based on laboratory orders, prescriptions, and duration of hospital stay, and acute onset without prior liver disease. Prognostic factors were identified using Cox regression analysis. 2) For microscopic cross-sectional observational study, a human explant liver from acute HBV infection was examined for immunohistochemical expression of progenitors [marker: CK19, epithelial cell adhesion molecule (EpCAM)], differentiation [NUMB (an inhibitor of the Notch pathway), carbamoyl phosphate synthetase 1 (CPS-1, urea cycle enzyme), HNF4α, and HNF1β], and proliferation (Ki-67). 3) For in vitro study, human hepatocytes (1 x 106 cells) isolated from unused donor livers were incubated at 37°C for 30 min with ICG (0-2 mg/ml) in both cell suspension and on collagen-coated culture plates. Cells were then incubated in medium without ICG for 3 h with supernatants collected at 1, 2, and 3 h for measurement of ICG release. Viability of cells was determined by trypan blue exclusion, MTT (mitochondrial dehydrogenase activity) and SRB (cell attachment) assays. HepG2 cells were also used as comparison. 4) For animal study, acute hepatic injury was induced in Sprague-Dawley rats with D-galactosamine. Hepatocytes (1 x 107/ml) were infused intraportally over 30, 70, or 100 seconds to study early engraftment (2 days) and repopulation (7 days). Results 1) For population study, during the study period, 218 eligible cases were identified from 28,078 potential eligible ALF patients. The incidence was 80.2 per million person-years in average and increased with age. The mean age was 57.9±17.1 years and median survival was 171 days. The most common etiologies were viral (45.4%, mainly hepatitis B virus) and alcohol/toxin (33.0%). Independent prognostic factors included alcohol consumption (HR 1.67 [1.01-2.77]), malignancy (HR 2.90 [1.92-4.37]), frequency of check-ups per week for total bilirubin (HR 1.57 [1.40-1.76]), sepsis (HR 1.85 [1.20-2.85]), and use of hemodialysis/hemofiltration (HR 2.12 [1.15-3.9]) and proton pump inhibitor (HR 0.94 [0.90-0.98]). Among the 130 patients who survived ≥90 days, 66 (50.8%) were complicated by liver cirrhosis. Eight (3.7%) were referred for liver transplantation evaluation, but only one received transplantation and survived. 2) For cross-sectional study, histological examination of the explant liver showed submassive necrosis and prominent ductular reaction. The road of hepatocyte differentiation was nicely shown from the bipotential progenitor cells (thick stained, small cell size, high nuclear-cytoplasm ratio) and gradually spirally spreading outward to form daughter intermediate hepatocytes (light stained, larger cell size, lower nuclear-cytoplasm ratio). These differentiating cells did not proliferate actively, and express EpCAM and transition of NUMB and CPS-1. 3) For in vitro study, ICG was taken up and secreted by hepatocytes with the release reaching a plateau level soon after 1 hour. Concentrations of ICG above 1.0 mg/ml, had toxic effects on hepatocytes. Hepatocytes incubated with 1.0 mg/ml ICG had higher mitochondrial dehydrogenase activity compared to 0.5 mg/ml ICG or control (0.025 ± 0.0004 v.s 0.019 ± 0.0008 or 0.020 ± 0.002, P < 0.05). Incubation of HepG2 cells with ICG reduced albumin production (98.9 ± 0.02, 66.6 ± 0.05, 39.1 ± 0.4 ng/ml for control, 0.5 mg/ml, and 1.0 mg/ml ICG respectively) and also decreased [3H]-thymidine incorporation in a dose-response manner. 4) For animal study, three groups had significant difference in hepatocyte engraftment (P = 0.018) and repopulation efficiencies (P = 0.037) and infusion over 70 seconds produced superior outcomes. After the 70-second infusion, the transplanted cells immediately transmigrated the sinusoidal endothelial layer and rarely accumulated in the portal venules, with improved liver function significantly. The mean first peak pressures, without significant difference, were 14.8 ± 6.5, 17.7 ± 3.7, and 13.6 ± 3.0 mmHg in the 30, 70, and 100-second groups, respectively. Conclusion ALF in Taiwan is mainly due to viral infection. Patients with malignancy and alcohol exposure have worst prognosis. The use of proton pump inhibitor is associated with improved survival. Half of the ALF survivors have liver cirrhosis. Prominent ductular reaction with at-least partially functional hepatocyte differentiation did not guarantee successful regeneration in acute liver failure and there is demand left for hepatocyte transplantation. With further refinement of ICG could be used to develop a rapid assay for assessment of the function of isolated human hepatocytes. Differential hepatocyte transfusion rate contribute to accelerated early engraftment and repopulation in rats with acute liver injury. These proof-of-concept findings are of clinical significance because they are easy to translate into practice. Further studies are needed for improvement of hepatocyte transplantation for ALF in Taiwan, albeit some problems solved.
Subjects
acute liver failure
prognosis
population
hepatocyte
progenitor cell
indocyanine green (ICG)
hepatocyte transplantation
engraftment
repopulation
SDGs
Type
thesis
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