Adult Hepatocytes Research Articles

Proof-of-Concept Gene Editing for the Murine Model of Inducible Arginase-1 Deficiency

Arginase-1 deficiency in humans is a rare genetic disorder of metabolism resulting from a loss of arginase-1, leading to impaired ureagenesis, hyperargininemia and neurological deficits. Previously, we generated a tamoxifen-inducible arginase-1 deficient mouse model harboring a deletion of Arg1 exons 7 and 8 that leads to similar biochemical defects, along with a wasting phenotype and death within two weeks. Here, we report a strategy utilizing the Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system in conjunction with piggyBac technology to target and reincorporate exons 7 and 8 at the specific Arg1 locus in attempts to restore the function of arginase-1 in induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iHLCs) and macrophages in vitro. While successful gene targeted repair was achieved, minimal urea cycle function was observed in the targeted iHLCs compared to adult hepatocytes likely due to inadequate maturation of the cells. On the other hand, iPSC-derived macrophages expressed substantial amounts of “repaired” arginase. Our studies provide proof-of-concept for gene-editing at the Arg1 locus and highlight the challenges that lie ahead to restore sufficient liver-based urea cycle function in patients with urea cycle disorders.

Higher Serum Alanine Transaminase Levels in Male Urokinase-Type Plasminogen Activator-Transgenic Mice Are Associated With Improved Engraftment of Hepatocytes but not Liver Sinusoidal Endothelial Cells.

The effects of sex on the degree of liver damage and human cell engraftment were investigated in immunodeficient urokinase-type plasminogen activator-transgenic (uPA-NOG) mice. Liver damage, measured by serum alanine transaminase (ALT) levels, was compared in male and female uPA-NOG mice of different ages. Male mice had significantly higher ALT levels than females with a median of 334 versus 158 U/L in transgenic homozygous mice, respectively. Mice were transplanted with human adult hepatocytes or fetal liver cells and analyzed for any correlation of engraftment of hepatocytes, liver sinusoidal endothelial cells (LSECs), and hematopoietic cells with the degree of liver damage. Hepatocyte engraftment was measured by human albumin levels in the mouse serum. Higher ALT levels correlated with higher hepatocyte engraftment, resulting in albumin levels in male mice that were 9.6 times higher than in females. LSEC and hematopoietic cell engraftment were measured by flow cytometric analysis of the mouse liver and bone marrow. LSEC and hematopoietic engraftment did not differ between male and female transplant recipients. Thus, the sex of uPA-NOG mice affects the degree of liver damage, which is reflected in the levels of human hepatocyte engraftment. However, the high levels of LSEC engraftment observed in uPA-NOG mice are not further improved among male mice, suggesting that a lower threshold of liver damage is sufficient to enhance endothelial cell engraftment. Previously described sex differences in human hematopoietic stem cell engraftment in immunodeficient mice were not observed in this model.

Generation of Canine Induced Extraembryonic Endoderm-Like Cell Line That Forms Both Extraembryonic and Embryonic Endoderm Derivatives.

Extraembryonic endoderm (XEN) cells are stem cell lines derived from primitive endoderm cells of inner cell mass in blastocysts. These cells have self-renewal properties and differentiate into visceral endoderm (VE) and parietal endoderm (PE) of the yolk sac. Recently, it has been reported that XEN cells can contribute to fetal embryonic endoderm, and their unique potency has been evaluated. In this study, we have described the induction and characterization of new canine stem cell lines that closely resemble to XEN cells. These cells, which we designated canine induced XEN (ciXEN)-like cells, were induced from canine embryonic fibroblasts by introducing four transgenes. ciXEN-like cells expressed XEN markers, which could be maintained over 50 passages in N2B27 medium supplemented with inhibitors of mitogen-activated protein kinase p38 and transforming growth factor-beta 1. Our ciXEN-like cells were maintained without transgene expression and exhibited upregulated expression of VE and PE markers in feeder-free conditions. The cells differentiated from ciXEN-like cells using a coculture system showed multiple nuclei and expressed albumin protein, similar to characteristics of hepatocytes. Furthermore, these cells expressed the adult hepatocyte marker, CYP3A4. Interestingly, these cells also formed a net structure expressing the bile epithelium capillary marker, multidrug resistance-associated protein 2. Thus, we have demonstrated the induction of a new canine stem cell line, ciXEN-like cells, which could form an embryonic endodermal cell layer. Our ciXEN-like cells may be a helpful tool to study the canine embryo development and represent a promising cell source for proceeding human and canine regenerative medicine.

Rat Liver Repopulation by Transplanted Late Gestation Fetal Hepatocytes

Hepatocyte transplantation has the potential to be an effective therapy for liver failure. However, the application of this therapy has been hampered by identification of an appropriate cell population and limited understanding of the factors accounting for the ability of cells to repopulate injured liver. Late gestation (E19) fetal hepatocyte proliferation in the rat is mitogen‐independent in vivo and in vitro. Fetal hepatocytes also differ from adult hepatocytes with regard to ribosome biogenesis, translation control, gene expression and cell cycle regulation. Our current studies are based on the hypothesis that histone posttranslational modifications (PTMs) acting through effects on chromatin structure account for the fetal hepatocyte signaling phenotype and that this phenotype will result in a selective growth advantage for fetal cells in the injured adult liver microenvironment. Late gestation fetal hepatocytes were isolated using antibodies against hepatic and ductal cell surface markers. The engraftment and repopulating ability of these cells was assessed using the mitomycin C dipeptidyl peptidase IV (DPPIV) transplant model. Liver harvested at 4 and 42 weeks posttransplantation contained well defined donor hepatocyte and endothelial colonies. Morphologic and phenotypic analysis using antibodies against well characterized markers revealed the formation of ductal colonies by 10 months. The average size of the donor colonies increased so that by 10 months up to 30% of the liver was repopulated by donor‐derived cells. Characterization of the donor cells pretransplant revealed that a large subpopulation of the hepatocytes also expressed ductal markers. Transcriptomic analysis revealed that these cells represent a distinct subpopulation of less well differentiated hepatocytes. Mass spectrometry‐based profiling of 77 modifications across 26 lysines on Histones H1, H2A, H3 & H4 revealed 23 significant changes including differential methylation of H3K27, H3K36 and H4K20 as well as acetylation of H3K4 comparing fetal to adult hepatocytes. In contrast, only 5 sites were significant comparing the two subpopulations of fetal hepatocytes. In conclusion, late gestation fetal hepatocytes are capable of extensive long‐term liver repopulation. These cells possess a histone PTM profile that is distinct from that of adult hepatocytes and may contribute to the maintenance of a fetal hepatocyte phenotype upon transplantation.Support or Funding InformationThe studies were supported by NIH grants R01DK100301 and P41GM108569. Support was also provided by the Rhode Island Hospital Department of Pediatrics.

The effects of CTRP3 on lipid oxidation in adult mouse primary hepatocytes

C1q Tumor Necrosis Factor‐Related Protein (CTRP3) is an adipose tissue‐derived secreted factor, or adipokine, that contributes to the processes of metabolism and inflammation. Our previous work has demonstrated that CTRP3 inhibits hepatic lipid biogenesis, in vivo and in vitro. Additionally, recombinant CTRP3 treatment induced the up‐regulation of lipid oxidation in rat H4IIE hepatoma cell line (a liver‐like cell line). However, CTRP3‐induced increase to lipid utilization in primary hepatocytes has not been examined. Although the H4IIE can provide insight into hepatic metabolism, by design immortalized cell lines function very differently than the tissue from which they were derived. Therefore, we isolated primary hepatocytes from wildtype (WT) and transgenic mice which overexpress CTRP3 (CTRP3 Tg), to test the effects of elevated CTRP3 on hepatocyte metabolism (primary hepatocytes, ex vivo, mirror the physiological metabolism of in vivo hepatocytes). Real‐time oxygen consumption rates (OCR) of hepatocytes were recorded by Extracellular Flux Analyzer. Protein expression was measured by Western Blot. We found that OCR were unchanged between WT and CTRP3 Tg mice under standard conditions, after the addition of 100 μM palmitic acid, however, hepatocytes from CTRP3 Tg mice had a significant ~45% increase in OCR consistent with a shift of respiratory substrate from glucose to fatty acid. Further, primary hepatocytes from CTRP3 Tg mice had a 50% increase in Akt phosphorylation (Ser473) in response to insulin. In conclusion, this study confirmed that CTRP3 increases hepatic lipid oxidation, ex vivo, and has provided more evidence to support that lipid oxidation increased by CTRP3 relies on b‐oxidation of fatty acid in primary hepatocytes.Support or Funding InformationThis research was supported in part National Institute On Alcohol Abuse And Alcoholism of the National Institutes of Health under Award Number R03AA023612 and East Tennessee State University Research Development Committee (E82262). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

CFD assessment of the effect of convective mass transport on the intracellular clearance of intracellular triglycerides in macrosteatotic hepatocytes.

Donor livers available to transplant for patients with end-stage liver disease are in severe shortage. One possible avenue to expand the donor pool is to recondition livers that would be otherwise discarded due to excessive fat content. Severely steatotic livers (also known as fatty livers) are highly susceptible to ischemia-reperfusion injury and as a result, primary liver non-function post-transplantation. Prior studies in isolated perfused rat livers suggest that "defatting" may be possible in a timeframe of a few hours; thus, it is conceivable that fatty liver grafts could be recovered by machine perfusion to clear stored fat from the organ prior to transplantation. However, studies using hepatoma cells and adult hepatocytes made fatty in culture report that defatting may take several days. Because cell culture studies were done in static conditions, we hypothesized that the defatting kinetics are highly sensitive to flow-mediated transport of metabolites. To investigate this question, we experimentally evaluated the effect of increasing flow rate on the defatting kinetics of cultured HepG2 cells and developed an in silico combined reaction-transport model to identify possible rate-limiting steps in the defatting process. We found that in cultured fatty HepG2 cells, the time required to clear stored fat down to lean control cells can be reduced from 48 to 4-6h by switching from static to flow conditions. The flow required resulted in a fluid shear of .008Pa, which did not adversely affect hepatic function. The reaction-transport model suggests that the transport of L-carnitine, which is the carrier responsible for taking free fatty acids into the mitochondria, is the key rate-limiting process in defatting that was modulated by flow. Therefore, we can ensure higher levels of L-carnitine uptake by the cells by choosing flow rates that minimize the limiting mass transport while minimizing shear stress.

Stepwise reprogramming of liver cells to a pancreas progenitor state by the transcriptional regulator Tgif2

The development of a successful lineage reprogramming strategy of liver to pancreas holds promises for the treatment and potential cure of diabetes. The liver is an ideal tissue source for generating pancreatic cells, because of its close developmental origin with the pancreas and its regenerative ability. Yet, the molecular bases of hepatic and pancreatic cellular plasticity are still poorly understood. Here, we report that the TALE homeoprotein TGIF2 acts as a developmental regulator of the pancreas versus liver fate decision and is sufficient to elicit liver-to-pancreas fate conversion both ex vivo and in vivo. Hepatocytes expressing Tgif2 undergo extensive transcriptional remodelling, which represses the original hepatic identity and, over time, induces a pancreatic progenitor-like phenotype. Consistently, in vivo forced expression of Tgif2 activates pancreatic progenitor genes in adult mouse hepatocytes. This study uncovers the reprogramming activity of TGIF2 and suggests a stepwise reprogramming paradigm, whereby a ‘lineage-restricted’ dedifferentiation step precedes the identity switch.

Reprogrammed Cell?based Therapy for Liver Disease: From Lab to Clinic

A large number of patients are affected by liver dysfunction worldwide. Liver transplantation is the only efficient treatment in a variety of enduring liver disorders including inherent and end-stage liver diseases. The generation of human functional hepatocytes in high quantities for liver cell therapy is an important goal for ongoing therapies in regenerative medicine. Reprogrammed cells are considered as a promising and unlimited source of hepatocytes, mainly because of their expected lack of immunogenicity and minimized ethical concerns in clinical applications. Despite gained advances in the reprogramming of somatic cells to functional hepatocytes in vitro, production of primary adult hepatocytes that can proliferate in vivo still remains inaccessible. As part of efforts toward translation of cell reprogramming science into clinical practice, more careful cell selection strategies should be integrated into improvement of dedifferentiation and redifferentiation protocols, especially in precision medicine where gene correction is needed. Furthermore, advances in cellular reprogramming highlight the need for developing and evaluating novel standards addressing clinical research interests in this field.

Pathology of intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCC) is a primary carcinoma of the liver with increasing significance and major pathogenic, clinical and therapeutic challenges. Classically, it arises from malignant transformation of cholangiocytes bordering small portal bile duct (BD) to second-order segmental large BDs. It has three major macroscopic growth pattern [mass-forming (MF), periductal infiltrative (PI), and intraductal growth (IG)] and histologically is a desmoplastic stroma-rich adenocarcinoma with cholangiocyte differentiation. Recent data pointed out noteworthy degree of heterogeneity in regards of their epidemiology and risk factors, pathological and molecular features, pathogenesis, clinical behaviors and treatment. Notably, several histological variants are described and can coexist within the same tumor. Several different cells of origin have also been depicted in a fraction of iCCs, amongst which malignant transformation of ductules, of hepatic stem/progenitor cells, of periductal glands or through oncogenic reprogramming of adult hepatocytes. A degree of pathological overlap with hepatocellular carcinoma (HCC) may be observed in a portion of iCC. A series of precursor lesions are today characterized and emphasize the existence of a multistep carcinogenesis process. Overall, these new data have brought up in proposal of new histological or molecular classifications, which could soon replace current anatomic-based classification and could have major impact on establishment of prognosis and on development of novel target treatment approaches.

A transcriptomic study suggesting human iPSC-derived hepatocytes potentially offer a better in vitro model of hepatotoxicity than most hepatoma cell lines.

Hepatocytes derived from human induced pluripotent stem cells (iPSCs) hold great promise as an in vitro liver model by virtue of their unlimited long-term supply, stability and consistency in functionality, and affordability of donor diversity. However, the suitability of iPSC-derived hepatocytes (iPSC-Heps) for toxicology studies has not been fully validated. In the current study, we characterized global gene expression profiles of iPSC-Heps in comparison to those of primary human hepatocytes (PHHs) and several human hepatoma cell lines (HepaRG, HuH-7, HepG2, and HepG2/C3A). Furthermore, genes associated with hepatotoxicity, drug-metabolizing enzymes, transporters, and nuclear receptors were extracted for more detailed comparisons. Our results showed that iPSC-Heps correlate more closely to PHHs than hepatoma cell lines, suggesting that iPSC-Heps had a relatively mature hepatic phenotype that more closely resembles that of adult hepatocytes. HepaRG was the sole exception but nonetheless suffers from lack of donor diversity and poor prediction of hepatotoxicity. The effects of sex differences and DMSO treatment on gene expression of the cellular models were also investigated. Overall, the results presented in the current study suggest that iPSC-Heps represent a reproducible source of human hepatocytes and a promising in vitro model for hepatotoxicity evaluation. Further studies are needed to develop a robust protocol for hepatocyte differentiation towards a more mature adult phenotype.

Preparation of Decellularized Liver Scaffolds and Recellularized Liver Grafts.

Severe liver disease is the 12th leading cause of death in the USA, with organ transplantation often being the only viable option for treatment. However, due to the shortage of viable donor livers, it is estimated that over 1200 patients died in 2015 while waiting for liver transplantation. This highlights the need for alternative sources of viable organs. In this study, we describe a method that provides the groundwork for the development of functional liver grafts. The approach described here is for removal of cells from intact livers and subsequently repopulating them with functional liver cells. Briefly, rat livers are harvested and subjected to a series of perfusion decellularization steps using an anionic detergent such that an intact decellularized liver matrix (DLM) scaffold with preserved vascular architecture is obtained. Further, we describe methods to recellularize DLM scaffolds with adult primary hepatocytes, creating a liver graft that exhibits hepatic functions in vitro.

THU-397 - Age-dependent glycosylation of the sodium taurocholate co-transporte polypeptide: from neonates to adult human primary hepatocytes

THU-397 - Age-dependent glycosylation of the sodium taurocholate co-transporte polypeptide: from neonates to adult human primary hepatocytes

Isolation and cultivation of adult primary bovine hepatocytes from abattoir derived liver.

The aim was to establish a cell culture of adult primary bovine hepatocytes obtained from liver following slaughter and to cultivate the cells in a sandwich culture. Cells and medium samples were taken after separation of cells (day 0), during monolayer (days 1, 2 and 3) and during sandwich culture (days 1, 2, 3, 4, 7, 10 and 14). The mRNA expression of BAX, BCL2L, FAS, IGF-1 and GHR1A was measured as well as urea and LDH. Hepatocytes were obtained by using a two-step collagenase perfusion and were purified thereafter by density gradient centrifugation. The viability was 68.2 ± 9.5 %. In sandwich culture, cells have a typical polygonal hepatocyte-like shape, build cell-cell contacts, and show irregularity of cell borders suggesting bile canaliculi generation. The BAX mRNA expression increased on day 1 as well but decreased steadily until day 3 and remained constant for 14 days. Urea- and LDH-concentrations increased from day 4 to day 7. In conclusion, we found that it is possible to gather viable primary hepatocytes from adult bovine liver after slaughter, and that cells gathered this way show typical morphologies, urea-production and low LDH-leakage especially at day 4 in a sandwich system.

Discarded Livers Find a New Life: Engineered Liver Grafts Using Hepatocytes Recovered From Marginal Livers.

Treatment for end-stage liver failure is restricted by the critical shortage of donor organs; about 4000 people die in the USA while waiting for a transplantable organ. This situation has been a major driving force behind the rise of tissue engineering to build artificial tissues/organs. Recent advancements in creating transplantable liver grafts using decellularized liver scaffolds bring the field closer to clinical translation. However, a source of readily available and highly functional adult hepatocytes in adequate numbers for regenerative liver therapies still remains unclear. Here, we describe a new method to utilize discarded livers to make transplantable new liver grafts. We show that marginal donor livers damaged due to warm ischemia could be treated with machine perfusion to yield 39 million viable hepatocytes per gram of liver, similar to fresh livers, and these cells could be used to repopulate decellularized liver matrix (DLM) scaffolds to make transplantable liver grafts. The hepatocytes from recovered livers sustained their characteristic epithelial morphology while they exhibited slightly lower protein synthesis functions both in plate cultures and in recellularized liver grafts. The dampened protein synthesis was attributed to residual endoplasmic reticulum stress found in recovered cells. The results here represent a unique approach to reengineer transplantable liver grafts solely from discarded organs.

Ductular reaction-on-a-chip: Microfluidic co-cultures to study stem cell fate selection during liver injury.

Liver injury modulates local microenvironment, triggering production of signals that instruct stem cell fate choices. In this study, we employed a microfluidic co-culture system to recreate important interactions in the liver stem cell niche, those between adult hepatocytes and liver progenitor cells (LPCs). We demonstrate that pluripotent stem cell-derived LPCs choose hepatic fate when cultured next to healthy hepatocytes but begin biliary differentiation program when co-cultured with injured hepatocytes. We connect this fate selection to skewing in production of hepatocyte growth factor (HGF) and transforming growth factor (TGF)-β1 caused by injury. Significantly, biliary fate selection of LPCs was not observed in the absence of hepatocytes nor did it happen in the presence of TGF-β inhibitors. Our study demonstrates that microfluidic culture systems may offer an interesting new tool for dissecting cellular interactions leading to aberrant stem cell differentiation during injury.

Efficient recellularisation of decellularised whole-liver grafts using biliary tree and foetal hepatocytes.

A whole-organ regeneration approach, using a decellularised xenogeneic liver as a scaffold for the construction of a transplantable liver was recently reported. Deriving suitable scaffolds was the first step towards clinical application; however, effective recellularisation remains to be achieved. This report presents a strategy for the improvement of the recellularisation process, using novel cell-seeding technique and cell source. We evaluated recellularised liver grafts repopulated through the portal vein or the biliary duct with mice adult hepatocytes or E14.5 foetal hepatocytes. More than 80% of the cells seeded through the biliary tree entered the parenchyma beyond the ductule-lining matrix barrier and distributed throughout the liver lobule. In contrast, about 20% of the cells seeded through the portal tree entered the parenchyma. The gene expression levels of foetal hepatocyte albumin, glucose 6-phosphatase, transferrin, cytokeratin 19, and gamma-glutamyl transpeptidase were increased in three-dimensional cultures in the native liver-derived scaffolds, and the activation of liver detoxification enzymes and formation of biliary duct-like structures were supported. The metabolic functions of liver grafts recellularised with different cell types were similar. These results suggest that biliary tree cell-seeding approach is promising, and that liver progenitor cells represent a good cell source candidate.

PDGF Facilitates Direct Lineage Reprogramming of Hepatocytes to Functional β-Like Cells Induced by Pdx1 and Ngn3.

Islet transplantation has been proven to be an effective treatment for patients with type 1 diabetes, but a lack of islet donors limits the use of transplantation therapies. It has been previously demonstrated that hepatocytes can be converted into insulin-producing β-like cells by introducing pancreatic transcription factors, indicating that direct hepatocyte reprogramming holds potential as a treatment for diabetes. However, the efficiency at which functional β-cells can be derived from hepatocyte reprogramming remains low. Here we demonstrated that the combination of Pdx1 and Ngn3 can trigger reprogramming of mouse and human liver cells to insulin-producing cells that exhibit the characteristics of pancreatic β-cells. Treatment with PDGF-AA was found to facilitate Pdx1 and Ngn3-induced reprogramming of hepatocytes to β-like cells with the ability to secrete insulin in response to glucose stimulus. Importantly, this reprogramming strategy could be applied to adult mouse primary hepatocytes, and the transplantation of β-like cells derived from primary hepatocyte reprogramming could ameliorate hyperglycemia in diabetic mice. These findings support the possibility of developing transplantation therapies for type 1 diabetes through the use of β-like cells derived from autologous hepatocyte reprogramming.

MST1/MST2 Protein Kinases: Regulation and Physiologic Roles.

The MST1 and MST2 protein kinases comprise the GCK-II subfamily of protein kinases. In addition to their amino-terminal kinase catalytic domain, related to that of the Saccharomyces cerevisiae protein kinase Ste20, their most characteristic feature is the presence near the carboxy terminus of a unique helical structure called a SARAH domain; this segment allows MST1/MST2 to homodimerize and to heterodimerize with the other polypeptides that contain SARAH domains, the noncatalytic polypeptides RASSF1-6 and Sav1/WW45. Early studies emphasized the potent ability of MST1/MST2 to induce apoptosis upon being overexpressed, as well as the conversion of the endogenous MST1/MST2 polypeptides to constitutively active, caspase-cleaved catalytic fragments during apoptosis initiated by any stimulus. Later, the cleaved, constitutively active form of MST1 was identified in nonapoptotic, quiescent adult hepatocytes as well as in cells undergoing terminal differentiation, where its presence is necessary to maintain those cellular states. The physiologic regulation of full length MST1/MST2 is controlled by the availability of its noncatalytic SARAH domain partners. Interaction with Sav1/WW45 recruits MST1/MST2 into a tumor suppressor pathway, wherein it phosphorylates and activates the Sav1-bound protein kinases Lats1/Lats2, potent inhibitors of the Yap1 and TAZ oncogenic transcriptional regulators. A constitutive interaction with the Rap1-GTP binding protein RASSF5B (Nore1B/RAPL) in T cells recruits MST1 (especially) and MST2 as an effector of Rap1's control of T cell adhesion and migration, a program crucial to immune surveillance and response; loss of function mutation in human MST1 results in profound immunodeficiency. MST1 and MST2 are also regulated by other protein kinases, positively by TAO1 and negatively by Par1, SIK2/3, Akt, and cRaf1. The growing list of candidate MST1/MST2 substrates suggests that the full range of MST1/MST2's physiologic programs and contributions to pathophysiology remains to be elucidated.

Expression of sal-like 4 in primary hepatocellular carcinoma and its association with epithelial-mesenchymal transition

Objective: To investigate the expression of sal-like 4 (SALL4) in tissues of primary hepatocellular carcinoma (HCC) and its association with epithelial-mesenchymal transition (EMT) and clinicopathological characteristics. Methods: Immunohistochemistry and nucleic acid in situ hybridization were used to measure the mRNA expression of SALL4, epithelial cadherin (E-cadherin), vimentin, and Snail in 72 HCC samples, 2 fetal liver samples, and 2 normal adult liver tissue samples. Results: Strong expression of SALL4 was observed in hepatoblasts in fetal liver, but SALL4 expression was not observed in primitive hematopoietic cells and normal adult hepatocytes or biliary epithelial cells. In the HCC samples, the positive rate of SALL4 was 47.2% (34/72), showing focal positive nuclear staining. The HCC patients with microscopic microvascular tumor thrombus and portal vein tumor thrombus, a serum alpha-fetoprotein level of≥ 350 ng/ml, International Union Against Cancer (UICC) stage III+IV, and an age of < 46 years showed higher positive expression of SALL4 than those with no microscopic microvascular tumor thrombus or portal vein tumor thrombus, a serum alpha-fetoprotein level of < 350 ng/ml, UICC stage I+II, and an age of≥46 years. The HCC patients with positive SALL4 showed lower postoperative disease-free survival rate and overall survival rate than those with negative SALL4 (P < 0.05). Sixty percent of the patients with microvascular tumor thrombus (21/35) showed positive expression of SALL4. The positive rate of SALL4 was negatively correlated with E-cadherin (r = -0.434, P < 0.01), but positively correlated with vimentin and Snail (vimentin: r = 0.516, P < 0.01; Snail: r = 0.571, P < 0.01). Conclusion: In patients with primary HCC, the expression of SALL4 greatly affects EMT, which helps with the research on invasion and metastasis of liver cancer and prognostic evaluation.

Human Upcyte Hepatocytes: Characterization of the Hepatic Phenotype and Evaluation for Acute and Long-Term Hepatotoxicity Routine Testing

The capacity of human hepatic cell-based models to predict hepatotoxicity depends on the functional performance of cells. The major limitations of human hepatocytes include the scarce availability and rapid loss of the hepatic phenotype. Hepatoma cells are readily available and easy to handle, but are metabolically poor compared with hepatocytes. Recently developed human upcyte hepatocytes offer the advantage of combining many features of primary hepatocytes with the unlimited availability of hepatoma cells. We analyzed the phenotype of upcyte hepatocytes comparatively with HepG2 cells and adult primary human hepatocytes to characterize their functional features as a differentiated hepatic cell model. The transcriptomic analysis of liver characteristic genes confirmed that the upcyte hepatocytes expression profile comes closer to human hepatocytes than HepG2 cells. CYP activities were measurable and showed a similar response to prototypical CYP inducers than primary human hepatocytes. Upcyte hepatocytes also retained conjugating activities and key hepatic functions, e.g. albumin, urea, lipid and glycogen synthesis, at levels close to hepatocytes. We also investigated the suitability of this cell model for preclinical hepatotoxicity risk assessments using multiparametric high-content screening, as well as transcriptomics and targeted metabolomic analysis. Compounds with well-documented in vivo hepatotoxicity were screened after acute and repeated doses up to 1 week. The evaluation of complex mechanisms of cell toxicity, drug-induced steatosis and oxidative stress biomarkers demonstrated that, by combining the phenotype of primary human hepatocytes and the ease of handling of HepG2 cells, upcyte hepatocytes offer suitable properties to be potentially used for toxicological assessments during drug development.