Novel immortalized human fetal liver cell line, cBAL111, has the potential to differentiate into functional hepatocytes

BACKGROUND: A clonal cell line that combines both stable hepatic function and proliferation capacity is desirable for in vitro applications that depend on hepatic function, such as pharmacological or toxicological assays and bioartificial liver systems. Here we describe the generation and characterization of a clonal human cell line for in vitro hepatocyte applications. RESULTS: Cell clones derived from human fetal liver cells were immortalized by over-expression of telomerase reverse transcriptase. The resulting cell line, cBAL111, displayed hepatic functionality similar to the parental cells prior to immortalization, and did not grow in soft agar. Cell line cBAL111 produced urea, albumin, cytokeratin 18 and 19 and showed high glutathione S transferase pi mRNA levels. In contrast to hepatic cell lines NKNT-3 and HepG2, all hepatic functions were expressed in cBAL111, although there was considerable variation in their levels compared with primary mature hepatocytes. When transplanted in the spleen of immunodeficient mice, cBAL111 engrafted into the liver and partly differentiated into hepatocytes showing expression of human albumin and carbamoylphosphate synthetase without signs of cell fusion. CONCLUSION: This novel liver cell line has the potential to differentiate into mature hepatocytes to be used for in vitro hepatocyte applications

Hepatoblast and mesenchymal cell-specific gene-expression in fetal rat liver and in cultured fetal rat liver cells

The aim of this study was to determine whether passaged rat fetal liver cells are functional hepatoblasts. Hepatocyte/hepatoblast- and liver myofibroblast-gene-expressions were studied in adult and fetal rat liver tissues as well as in primary and passaged cultures of isolated rat fetal liver cells at both the mRNA and protein level. Desmin- and Alpha-Smooth Muscle Actin (SMA)-positive cells were located in the walls of liver vessels, whereas Desmin-positive/SMA-negative cells were distributed within the liver parenchyma. Primary cultures contained Prox1-positive hepatoblasts, Desmin/SMA-positive myofibroblasts and only a few Desmin-positive/SMA-negative cells. Albumin and alpha-fetoprotein (AFP) could be detected in the primary cultures and to a lesser extent after the first passage. The number of Desmin-positive/SMA-negative cells decreased with successive passage, such that after the second passage, only Desmin/SMA-positive cells could be detected. SMA-gene-expression increased during the passages, suggesting that myofibroblasts become the major cell population of fetal liver cell cultures over time. This observation needs to be taken into account, should passaged fetal liver cells be used for liver cell transplantation. Moreover it contradicts the concept of epithelial-mesenchymal transformation and suggests rather that selective overgrowth of mesenchymal cells occurs in culture

Liver Progenitor Cell Line HepaRG Differentiated in a Bioartificial Liver Effectively Supplies Liver Support to Rats with Acute Liver Failure

A major roadblock to the application of bioartificial livers is the need for a human liver cell line that displays a high and broad level of hepatic functionality. The human bipotent liver progenitor cell line HepaRG is a promising candidate in this respect, for its potential to differentiate into hepatocytes and bile duct cells. Metabolism and synthesis of HepaRG monolayer cultures is relatively high and their drug metabolism can be enhanced upon treatment with 2% dimethyl sulfoxide (DMSO). However, their potential for bioartificial liver application has not been assessed so far. Therefore, HepaRG cells were cultured in the Academic Medical Center bioartificial liver (AMC-BAL) with and without DMSO and assessed for their hepatic functionality in vitro and in a rat model of acute liver failure. HepaRG-AMC-BALs cultured without DMSO eliminated ammonia and lactate, and produced apolipoprotein A-1 at rates comparable to freshly isolated hepatocytes. Cytochrome P450 3A4 transcript levels and activity were high with 88% and 37%, respectively, of the level of hepatocytes. DMSO treatment of HepaRG-AMC-BALs reduced the cell population and the abovementioned functions drastically. Therefore, solely HepaRG-AMC-BALs cultured without DMSO were tested for efficacy in rats with acute liver failure (n = 6). HepaRG-AMC-BAL treatment increased survival time of acute liver failure rats ∼50% compared to acellular-BAL treatment. Moreover, HepaRG-AMC-BAL treatment decreased the progression of hepatic encephalopathy, kidney failure, and ammonia accumulation. These results demonstrate that the HepaRG-AMC-BAL is a promising bioartificial liver for clinical application

Comorbidity and survival in the pre-hospital and in-hospital phase after out-of-hospital cardiac arrest

Introduction: Cumulative disease burden may be associated with survival chances after out-of-hospital cardiac arrest (OHCA). The relative contributions of cumulative disease burden on survival rates at the pre-hospital and in-hospital phases of post-resuscitation care are unknown. Methods: The association between cumulative comorbidity burden as measured by the Charlson Comorbidity Index (CCI) and pre-hospital and in-hospital survival rates was studied using data (2010–2014) from a prospective OHCA registry in the Netherlands. The association between CCI and survival rate (overall survival [OHCA-hospital discharge], pre-hospital survival [OHCA-hospital admission] and in-hospital survival [hospital admission-hospital discharge]) was assessed using logistic regression analyses. The relative contributions of CCI on pre-hospital and in-hospital survival rates were determined using the Nagelkerke test. Results: We included 2510 OHCA patients aged ≥18y. CCI was significantly associated with overall survival rate (OR 0.71; 95%CI 0.61−0.83; P < 0.01). CCI was not associated with pre-hospital survival rate (OR 0.96; 95%CI 0.76–1.23; P = 0.92) whereas high CCI was significantly associated with low in-hospital survival rate (OR 0.41; 95%CI 0.27−0.62; P = 0.01). The relative contributions of CCI on pre-hospital and in-hospital survival were 1.1% and 8.1%, respectively. Conclusion: Pre-existing high comorbidity burden plays a modest role in reducing survival rate after OHCA, and only in the in-hospital phase. The present study offers data that may guide clinicians in discussing resuscitation options during advance care planning with patients with high comorbidity burden. This may be helpful in creating a patients’ informed choice

Comorbidity and survival in the pre-hospital and in-hospital phase after out-of-hospital cardiac arrest

Introduction: Cumulative disease burden may be associated with survival chances after out-of-hospital cardiac arrest (OHCA). The relative contributions of cumulative disease burden on survival rates at the pre-hospital and in-hospital phases of post-resuscitation care are unknown. Methods: The association between cumulative comorbidity burden as measured by the Charlson Comorbidity Index (CCI) and pre-hospital and in-hospital survival rates was studied using data (2010–2014) from a prospective OHCA registry in the Netherlands. The association between CCI and survival rate (overall survival [OHCA-hospital discharge], pre-hospital survival [OHCA-hospital admission] and in-hospital survival [hospital admission-hospital discharge]) was assessed using logistic regression analyses. The relative contributions of CCI on pre-hospital and in-hospital survival rates were determined using the Nagelkerke test. Results: We included 2510 OHCA patients aged ≥18y. CCI was significantly associated with overall survival rate (OR 0.71; 95%CI 0.61−0.83; P < 0.01). CCI was not associated with pre-hospital survival rate (OR 0.96; 95%CI 0.76–1.23; P = 0.92) whereas high CCI was significantly associated with low in-hospital survival rate (OR 0.41; 95%CI 0.27−0.62; P = 0.01). The relative contributions of CCI on pre-hospital and in-hospital survival were 1.1% and 8.1%, respectively. Conclusion: Pre-existing high comorbidity burden plays a modest role in reducing survival rate after OHCA, and only in the in-hospital phase. The present study offers data that may guide clinicians in discussing resuscitation options during advance care planning with patients with high comorbidity burden. This may be helpful in creating a patients’ informed choice

Evidence for Galalpha(1-3)Gal expression on primary porcine hepatocytes: implications for bioartificial liver systems

BACKGROUND/AIMS: To bridge acute liver failure (ALF) patients to orthotopic liver transplantation, several bioartificial liver (BAL) systems have been developed. The bio-component of most BAL systems consists mainly of porcine hepatocytes. Plasma or blood of ALF patients is perfused through the BAL thereby contacting porcine hepatocytes. Xenogeneic BAL systems may suffer from hyperacute rejection similar to whole-organ xenotransplants. Hyperacute rejection is mediated by antibodies directed against Galalpha(1-3)Gal, a carbohydrate structure present on most mammalian cells. Galalpha(1-3)Gal is produced by the enzyme alpha1,3-galactosyltansferase (alphaGal-T). Conflicting data have been published concerning Galalpha(1-3)Gal expression on hepatocytes in intact porcine liver. We investigated whether isolated porcine hepatocytes express Galalpha(1-3)Gal. METHODS: Immunofluorescence, flow cytometry, RT-PCR and enzyme activity assays were performed on freshly isolated and cultured porcine hepatocytes and liver biopsies. Anti-Galalpha(1-3)Gal antibodies were measured in plasma from patients treated with BAL by ELISA. RESULTS: Isolated porcine hepatocytes express (alphaGal-T) at low levels and Galalpha(1-3)Gal is present in low quantities on these cells, in contrast to hepatocytes in situ. Furthermore, IgG and IgM anti-Galalpha(1-3)Gal are depleted from the plasma of ALF patients during BAL treatment. CONCLUSIONS: Isolation and culture of porcine hepatocytes induce Galalpha(1-3)Gal expression, which may elicit immunological responses potentially compromising BAL functionalit