Human Fetal Hepatocytes Research Articles

Effective Transplantation of 2D and 3D Cultured Hepatocyte Spheroids Confirmed by Quantum Dot Imaging

AbstractLiver transplantation is the most effective treatment for patients with end‐stage liver diseases, but hepatocyte transplantation using spheroids is expected to become an alternative strategy owing to the limited number of liver donors. However, the effects of the transplantation route and spheroid size on cell dynamics and the organ‐specific accumulation of hepatocytes are not well characterized. In this study, the influence of three delivery routes (tail vein, portal vein, and spleen) and three spheroid sizes (50, 100, and 150 µm in diameter) on the accumulation of human fetal hepatocytes labeled with quantum dots in the liver and other organs (heart, lung, kidney, and spleen) of recipient mice is investigated. Ex vivo fluorescence imaging reveals that the accumulation of transplanted cells in the liver is highest for transplantation via the portal vein compared with other routes. The accumulation efficiency depends on spheroid size, and spheroids with a diameter of 50 µm display greater accumulation than that of spheroids of other sizes. It is concluded that hepatocyte transplantation via the portal vein using small spheroids of 50 µm in diameter may be effective for clinical applications.

Effect of Gestational Diabetes and Maternal Obesity on Fetal Programming—Are miRNAs Key Epigenetic Modifiers or Biomarkers of an Altered Intrauterine Milieu?

Background: Pregnancies complicated by gestational diabetes (GDM) or maternal obesity (MOB) induce abnormal fetal development that can lead to diabetes and obesity later in life with sex specific manifestations. Alterations in miRNA expression in GDM and MOB exposed offspring and effects on development have not been fully described. Hypothesis: Exposure to an aberrant intrauterine milieu complicated by GDM and MOB leads to changes in miRNA and target gene expression in human fetal liver. Methods: Candidate miRNA expression was measured in second trimester AF from women diagnosed with GDM via multiplex miRNA assay (Firefly Bioworks, Abcam). Gene targets of differentially expressed miRNAs were determined from TargetScan and pathway enrichment determined via Ingenuity Pathway Analysis (IPA). MiRNA and target gene expression (mRNA and protein) were measured in a separate cohort of 2nd trimester primary human fetal hepatocytes (PHFH) exposed to MOB via QPCR and western blot. All studies were approved by UPENN and TEMPLE University IRBs. Results: GDM exposed AF had significant increases in miRNAs 199a-3p, 503-5p, and 1268a (p<0.05) and sex specific analysis showed enrichment in miRNAs 378a-3p, 885-5p, and 7-1-3p in female offspring samples (p<0.05). IPA with gene targets of enriched miRNAs centered hepatic pathways. GDM exposed AF miRNA enrichment was confirmed in PHFH exposed to MOB for miRNAs 885-5p, 199-3p, 503-5p, and 7-1-3p (p<0.05). Female PHFH exposed to MOB were enriched for miRNAs 885-5p, 199-3p, 503-5p, and 7-1-3p (p<0.05) and corresponded to decreased expression of target genes ABCA1, PAK4. In male PHFHs, no miRNA expression changes were measured but there was increased expression of ABCA1 and PAK4 (p<0.05). Conclusion: Our data suggest sex specific changes in miRNA and target gene expression in PHFH may be one mechanism responsible for sex specific metabolic changes in offspring exposed to GDM and obesity in utero. Disclosure A. Joshi: None. L. Goetzl: None. S.E. Pinney: None.

Regulation of Cytosolic Sulfotransferases in Models of Human Hepatocyte Development.

Cytosolic sulfotransferases (SULTs) are expressed during early life and therefore metabolize endogenous and xenobiotic chemicals during development. Little is currently known about the regulation of individual SULTs in the developing human liver. We characterized SULT expression in primary cultures of human fetal hepatocytes and the HepaRG model of liver cell differentiation. SULT1A1 (transcript variants 1-4), SULT1C2, SULT1C4, SULT1E1, and SULT2A1 were the most abundant transcripts in human fetal hepatocytes. In HepaRG cells, SULT1B1, SULT1C2/3/4, and SULT1E1 mRNA levels increased during the transition from proliferation to confluency and then decreased as the cells underwent further differentiation. By contrast, SULT2A1 mRNA levels increased during differentiation, whereas SULT1A1 and SULT2B1 mRNA levels remained relatively constant. The temporal patterns of SULT1C2, SULT1E1, and SULT2A1 protein content were consistent with those observed at the mRNA level. To identify regulators of SULT expression, cultured fetal hepatocytes and HepaRG cells were treated with a panel of lipid- and xenobiotic-sensing receptor activators. The following effects were observed in both fetal hepatocytes and HepaRG cells: 1) liver X receptor activator treatment increased SULT1A1 transcript variant 5 levels; 2) vitamin D receptor activator treatment increased SULT1C2 and SULT2B1 mRNA levels; and 3) farnesoid X receptor activator treatment decreased SULT2A1 expression. Activators of aryl hydrocarbon receptor, constitutive androstane receptor, pregnane X receptor, and peroxisome proliferator-activated receptors produced additional gene-dependent effects on SULT expression in HepaRG cells. These findings suggest that SULT-regulating chemicals have the potential to modulate physiologic processes and susceptibility to xenobiotic stressors in the developing human liver.

Multi-color RGB marking enables clonality assessment of liver tumors in a murine xenograft model.

We recently introduced red-green-blue (RGB) marking for clonal cell tracking based on individual color-coding. Here, we applied RGB marking to study clonal development of liver tumors. Immortalized, non-tumorigenic human fetal hepatocytes expressing the human telomerase reverse transcriptase (FH-hTERT) were RGB-marked by simultaneous transduction with lentiviral vectors encoding mCherry, Venus, and Cerulean. Multi-color fluorescence microscopy was used to analyze growth characteristics of RGB-marked FH-hTERT in vitro and in vivo after transplantation into livers of immunodeficient mice with endogenous liver damage (uPA/SCID). After initially polyclonal engraftment we observed oligoclonal regenerative nodules derived from transplanted RGB-marked FH-hTERT. Some mice developed monochromatic invasive liver tumors; their clonal origin was confirmed both on the molecular level, based on specific lentiviral-vector insertion sites, and by serial transplantation of one tumor. Vector insertions in proximity to the proto-oncogene MCF2 and the transcription factor MITF resulted in strong upregulation of mRNA expression in the respective tumors. Notably, upregulated MCF2 and MITF expression was also observed in 21% and 33% of 24 human hepatocellular carcinomas analyzed. In conclusion, liver repopulation with RGB-marked FH-hTERT is a useful tool to study clonal progression of liver tumors caused by insertional mutagenesis in vivo and will help identifying genes involved in liver cancer.

In vitro evidences of epithelial to mesenchymal transition in low cell-density cultured human fetal hepatocytes

Culturing fetal hepatocytes in high cell-density allowed stabilization of the hepatocyte phenotype up to 8 weeks, including the maintenance of liver-specific functions. On the other hand, when cultured at low cell-density, fetal hepatocytes underwent morphological modifications and acquired fibroblastic morphology. Since a switch from E-cadherin to vimentin expression accompanied these changes, we hypothesized the occurrence of epithelial-to-mesenchymal transition when fetal hepatocytes were cultured at low cell-density. Changes in gene expressionsuch as up-regulation of fibrosis-related geneswere also observed, suggesting that the low cell-density culture system promoted the acquisition of a profibrotic phenotype in cultured hepatocytes. The origin of fibrogenic cells in the liver is not well known, and the role of hepatocytes as a source of fibrogenic cells is controversial. Therefore, we hypothesized that hepatocytes undergoing epithelial-to-mesenchymal transition could have a central role in liver fibrosis as a source of fibrogenic cells.To conclude, the high cell-density culture system could be a useful model for in vitro studies requiring long-term cultures of hepatocytes, such as the development of pharmaceutical drugs and mechanisms of viral infections. The low cell-density culture system may provide additional insights into the origin of fibrogenic cells in the liver, thus contributing to the development of novel therapeutic approaches.

Constitutive gp130 activation rapidly accelerates the transformation of human hepatocytes via an impaired oxidative stress response.

Pro-inflammatory signaling pathways, especially interleukin 6 (IL-6), and reactive oxygen species (ROS) promote carcinogenesis in the liver. In order to elucidate the underlying oncogenic mechanism, we activated the IL-6 signal transducer glycoprotein 130 (gp130) via stable expression of a constitutively active gp130 construct (L-gp130) in untransformed telomerase-immortalized human fetal hepatocytes (FH-hTERT). As known from hepatocellular adenomas, forced gp130 activation alone was not sufficient to induce malignant transformation. However, additional challenge of FH-hTERT L-gp130 clones with oxidative stress resulted in 2- to 3-fold higher ROS levels and up to 6-fold more DNA-double strand breaks (DSB). Despite increased DNA damage, ROS-challenged FH-hTERT L-gp130 clones displayed an enhanced proliferation and rapidly developed colony growth capabilities in soft agar. As driving gp130-mediated oncogenic mechanism, we detected a decreased expression of antioxidant genes, in particular glutathione peroxidase 3 and apolipoprotein E, and an absence of P21 upregulation following ROS-conferred induction of DSB. In summary, an impaired oxidative stress response in hepatocytes with gp130 gain-of-function mutations, as detected in dysplastic intrahepatic nodules and hepatocellular adenomas, is one of the central oncogenic mechanisms in chronic liver inflammation.

Arsenic (As) and Copper (Cu) Serve as Cofactors to Produce Hepatotoxicity With Mitochondrial Damage, Double Strand DNA Breaks and Disruption of ATM Signaling

Despite increasing exposure of populations to As the role of cofactors in organ toxicity, e.g., reactive Cu ions, is poorly defined. Since As and Cu ions may combine to increase oxphos events and processes directing mitochondrial and cellular DNA damage, we studied metal toxicity in cultured adult and fetal human hepatocytes with IC50 concentrations of As in MTT assays. In even nontoxic conditions by itself Cu dose‐dependently worsened As toxicity. This worsening included alterations related to oxphos activity with mitochondrial and nuclear perturbations (JC‐1 dye assays, acridine orange nuclear stain, 8‐OHdG stain), double strand DNA breaks (Comet assay, γH2AX stain), and replicative stress including loss of cycling cells and/or arrest in G2/M (Ki‐67staining, flow cytometry). In addition, verapamil‐sensitive side populations denoting stem cells were depleted by As toxicity. Use of cells stably transduced lentivirally to express hATM promoter construct with tdT reporter showed that Cu exacerbated cellular DNA damage by perturbing ATM signaling pathways. To further establish whether Cu accumulation may also worsen As toxicity in vivo, we studied Atp7b−/− mice that model Wilson's disease (WD) and accumulate Cu in liver due to absence of hepatobiliary Cu excretion, along with their healthy littermates. After As was given i.p. for 10d, liver necrosis increased significantly in Atp7b−/− mice (H&E tissue staining, serum hyaluronic acid levels). Moreover, we noted increased ATM pathway‐dependent DNA strand breaks (Comet assay, γH2AX stain). This As and Cu toxicity was ameliorated in human hepatocytes cultured with an anti‐oxidant, indole‐3 propionic acid (IPA), as shown by MTT, JC‐1 and hATM promoter assays. Similarly, IPA decreased As toxicity in Atp7b−/− mice.ConclusionsCu ions markedly exacerbated As toxicity with mitochondrial and ATM pathway‐regulated DNA damage. These findings will help understand how toxic metals may alter physiological processes and contribute additionally in human diseases, e.g., WD with Cu toxicosis.

Effects of macroporous hydroxyapatite carriers on the growth and function of human hepatoblasts derived from fetal hepatocytes

Improvement of three-dimensional (3D) culture conditions, including substrates for cell growth, is needed for various cell-based applications. In this study, we developed hydroxyapatite (HAp) macroporous carriers having several pore size distributions and tried to obtain the findings about the effective pore sizes for the growth and function of hepatoblasts derived from human fetal hepatocytes. Cellular CYP3A4 activity was significantly enhanced when 20% HAp macroporous carrier was used, reaching 1.49±0.28pmol/10(6)cells/min of benzyloxyresorufin-O-dealkylation activity, which is comparable to that of primary human hepatocytes from livers of adult donors. Analysis of the pore size (the radius of curvature) distribution of each HAp carrier using a 3D-electron beam surface roughness analyzer revealed two peaks of pore size distribution at 30-40μm and 70-80μm, respectively. Thirty-five percent of the pores in the 20% carrier had a size distribution within 50-80μm. Especially, pores of 70-80μm were more abundant in the 20% HAp carrier than in the 10% and 30% HAp carriers. These results suggested that a HAp carrier with the pore size distribution of 50-80μm might be effective for cell growth and function in human hepatoblasts derived from fetal hepatocytes.

SIRT1 Disruption in Human Fetal Hepatocytes Leads to Increased Accumulation of Glucose and Lipids.

There are unprecedented epidemics of obesity, such as type II diabetes and non-alcoholic fatty liver diseases (NAFLD) in developed countries. A concerning percentage of American children are being affected by obesity and NAFLD. Studies have suggested that the maternal environment in utero might play a role in the development of these diseases later in life. In this study, we documented that inhibiting SIRT1 signaling in human fetal hepatocytes rapidly led to an increase in intracellular glucose and lipids levels. More importantly, both de novo lipogenesis and gluconeogenesis related genes were upregulated upon SIRT1 inhibition. The AKT/FOXO1 pathway, a major negative regulator of gluconeogenesis, was decreased in the human fetal hepatocytes inhibited for SIRT1, consistent with the higher level of gluconeogenesis. These results indicate that SIRT1 is an important regulator of lipid and carbohydrate metabolisms within human fetal hepatocytes, acting as an adaptive transcriptional response to environmental changes.

Characterization of Liver-Specific Functions of Human Fetal Hepatocytes in Culture

This study was designed to assess liver-specific functions of human fetal liver cells proposed as a potential source for hepatocyte transplantation. Fetal liver cells were isolated from livers of different gestational ages (16-22 weeks), and the functions of cell preparations were evaluated by establishing primary cultures. We observed that 20- to 22-week-gestation fetal liver cell cultures contained a predominance of cells with hepatocytic traits that did not divide in vitro but were functionally competent. Fetal hepatocytes performed liver-specific functions at levels comparable to those of their adult counterpart. Moreover, exposure to dexamethasone in combination with oncostatin M promptly induced further maturation of the cells through the acquisition of additional functions (i.e., ability to store glycogen and uptake of indocyanine green). In some cases, particularly in cultures obtained from fetuses of earlier gestational ages (16-18 weeks gestation), cells with mature hepatocytic traits proved to be sporadic, and the primary cultures were mainly populated by clusters of proliferating cells. Consequently, the values of liver-specific functions detected in these cultures were low. We observed that a low cell density culture system rapidly prompted loss of the mature hepatocytic phenotype with downregulations of all the liver-specific functions. We found that human fetal liver cells can be cryopreserved without significant loss of viability and function and evaluated up to 1 year in storage in liquid nitrogen. They might, therefore, be suitable for cell banking and allow for the transplantation of large numbers of cells, thus improving clinical outcomes. Overall, our results indicate that fetal hepatocytes could be used as a cell source for hepatocyte transplantation. Fetal liver cells have been used so far to treat end-stage liver disease. Additional studies are needed to include these cells in cell-based therapies aimed to treat liver failure and inborn errors of metabolism.

Analysis of gene expressions and metabolic activities in fetal and postnatal human hepatocytes for clinical therapy

Analysis of gene expressions and metabolic activities in fetal and postnatal human hepatocytes for clinical therapy

Endogenous antiviral microRNAs determine permissiveness for hepatitis B virus replication in cultured human fetal and adult hepatocytes.

Superior cell culture models for hepatitis B virus (HBV) will help advance insights into host-virus interactions. To identify mechanisms regulating HBV replication, this study used cultured human HepG2 cells and adult or fetal hepatocytes transduced with adenoviral vector to express HBV upstream of green fluorescent protein. The vector efficiently transduced all cell types. In HepG2 cells, replicative viral intermediates, nucleocapsid-associated HBcAg, and HBsAg were expressed. However, in fetal or adult hepatocytes, pregenomic HBV RNA and viral RNAs were expressed, but nucleocapsid-associated HBcAg in cells or HBsAg in culture medium were absent, indicating interruptions in viral replication due to possible microRNA-related interference. MicroRNA profiling demonstrated that a large number of microRNAs with antiviral potential were differentially expressed in hepatocytes after culture. In transfection assays using HepG2 cells, candidate antiviral microRNAs, e.g., hsa-miR-24 or hsa-miR-638 decreased the levels of HBV transcripts or HBV gene products. Since candidate microRNAs could have targeted interferon response genes as an alternative explanation interferon signaling was examined. However, HBV replication in cultured hepatocytes was not restored despite successful inhibition of JAK1/2-STAT signaling by the inhibitor, ruxolitinib. Therefore, HBV was unable to complete replication in cultured hepatocytes due to expression of multiple antiviral microRNAs. This mechanism should help understand restrictions in HBV replication for developing HBV models in cultured cells while providing frameworks for pathophysiological studies of HBV replication in subsets of hepatocytes or stem/progenitor cells during hepatitis.

Chemokine-mediated robust augmentation of liver engraftment: a novel approach.

Effective repopulation of the liver is essential for successful clinical hepatocyte transplantation. The objective was to improve repopulation of the liver with human hepatocytes using chemokines. We used flow cytometry and immunohistochemistry assays to identify commonly expressed chemokine receptors on human fetal and adult hepatocytes. The migratory capacity of the cells to various chemokines was tested. For in vivo studies, we used a nude mouse model of partial hepatectomy followed by intraparenchymal injections of chemokine ligands at various concentrations. Human fetal liver cells transformed with human telomerase reverse transcriptase were used for intrasplenic cell transplantation. Repopulation and functionality were assessed 4 weeks after transplantation. The receptor CXCR3 was commonly expressed on both fetal and adult hepatocytes. Both cell types migrated efficiently toward corresponding CXC chemokine ligands 9, 10, and 11. In vivo, animals injected with recombinant chemokines showed the highest cell engraftment compared with controls (p<.05). The engrafted cells expressed several human hepatic markers such as cytokeratin 8 and 18 and albumin as well as transferrin, UGT1A1, hepatocyte nuclear factor (1α, 1β, and 4α), cytochrome CYP3A1, CCAAT/enhancer binding protein (α and β), and human albumin compared with controls. No inflammatory cells were detected in the livers at 4 weeks after transplantation. The improved repopulation of transplanted cells is likely a function of the chemokines to mediate cell homing and retention in the injured liver and might be an attractive strategy to augment repopulation of transplanted hepatocytes in vivo.

Potential Barriers to Human Hepatocyte Transplantation in MUP–uPAtg(+/+)Rag2-/-γC-/-Mice

Primary human fetal and adult hepatocytes have been considered feasible donor cell sources for cell transplantation. We compared the engraftment efficiencies between adult human, fetal human, and adult porcine hepatocytes after transplantation into MUP-uPA(tg(+/+))Rag2(-/-)γC(-/-)mice. Transplantation of adult human hepatocytes yielded a 1,000-fold higher serum albumin level compared to transplantation of fetal human hepatocytes, while transplantation of adult porcine hepatocytes resulted in a 100-fold higher serum albumin level than adult human hepatocytes. These results suggest that adult liver cells are superior to fetal liver cells for transplantation, and caution should be applied if porcine hepatocytes are used for preclinical studies as a proof of concept for human hepatocytes.

Optothermally responsive nanocomposite generating mechanical forces for cells enabled by few-walled carbon nanotubes.

We have designed and fabricated a nanocomposite substrate that can deliver spatially and temporally defined mechanical forces onto cells. This nanocomposite substrate comprises a 1.5-mm-thick near-infrared (NIR) mechanoresponsive bottom layer of few-walled carbon nanotubes (FWCNTs) that are uniformly distributed and covalently connected to thermally responsive poly(N-isopropylacrylamide) and an approximately 0.15-mm-thick cell-seeding top layer of collagen-functionalized poly(acrylic acid)-co-poly(N-isopropylacrylamide) that interpenetrates into the bottom layer. Covalent coupling of all the components and uniform distribution of FWCNTs lead to a large local mechanoresponse. As an example, 50% change in strain at the point of irradiation on the order of 0.05 Hz can be produced reversibly under NIR stimulation with 0.02 wt % FWCNTs. We have further demonstrated that the mechanical strain imposed by NIR stimulation can be transmitted onto cells. Human fetal hepatocytes change shape with no sign of detrimental effect on cell viability. To the best of our knowledge, this is the first demonstration of a nanocomposite platform that can generate fast and controlled mechanical force to actuate cells. Since the amplitude, location, and timing of force can be controlled remotely with NIR, the nanocomposite substrate offers the potential to provide accurately designed force sequences for tissue engineering.

Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes

Hepatocyte-like cells (HLCs), differentiated from pluripotent stem cells by the use of soluble factors, can model human liver function and toxicity. However, at present HLC maturity and whether any deficit represents a true fetal state or aberrant differentiation is unclear and compounded by comparison to potentially deteriorated adult hepatocytes. Therefore, we generated HLCs from multiple lineages, using two different protocols, for direct comparison with fresh fetal and adult hepatocytes. Protocols were developed for robust differentiation. Multiple transcript, protein and functional analyses compared HLCs to fresh human fetal and adult hepatocytes. HLCs were comparable to those of other laboratories by multiple parameters. Transcriptional changes during differentiation mimicked human embryogenesis and showed more similarity to pericentral than periportal hepatocytes. Unbiased proteomics demonstrated greater proximity to liver than 30 other human organs or tissues. However, by comparison to fresh material, HLC maturity was proven by transcript, protein and function to be fetal-like and short of the adult phenotype. The expression of 81% phase 1 enzymes in HLCs was significantly upregulated and half were statistically not different from fetal hepatocytes. HLCs secreted albumin and metabolized testosterone (CYP3A) and dextrorphan (CYP2D6) like fetal hepatocytes. In seven bespoke tests, devised by principal components analysis to distinguish fetal from adult hepatocytes, HLCs from two different source laboratories consistently demonstrated fetal characteristics. HLCs from different sources are broadly comparable with unbiased proteomic evidence for faithful differentiation down the liver lineage. This current phenotype mimics human fetal rather than adult hepatocytes.

Immortalization of Human Fetal Hepatocyte by Ectopic Expression of Human Telomerase Reverse Transcriptase, Human Papilloma Virus (E7) and Simian Virus 40 Large T (SV40 T) Antigen Towards Bioartificial Liver Support

Generation of genetically stable and non-tumoric immortalization cell line from primary cells would be enormously useful for research and therapeutic purposes, but progress towards this goal has so far been limited. It is now universal acceptance that immortalization of human fetal hepatocytes based on recent advances of telomerase biology and oncogene, lead to unlimited population doubling could be the possible source for bioartificial liver device. Immortalization of human fetal hepatocytes cell line by ectopic expression of human telomerase reverse transcriptase (hTERT), human papilloma virus gene (E7) and simian virus 40 large T (SV40 T) antigens is main goal of present study. We used an inducible system containing human telomerase and E7, both of which are cloned into responder constructs controlled by doxycycline transactivator. We characterized the immortalized human fetal hepatocyte cells by analysis of green fluorescent cells (GFP) positive cells using flow cytometry (FACs) cell sorting and morphology, proliferative rate and antigen expression by immunohistochemical analysis. In addition to we analysized lactate formation, glucose consumption, albumin secretion and urea production of immortalized human fetal hepatocyte cells. After 25 attempts for transfection of adult primary hepatocytes by human telomerase and E7 to immortalize them, none of the transfection systems resulted in the production of a stable, proliferating cell line. Although the transfection efficiency was more than 70% on the first day, the vast majority of the transfected hepatocytes lost their signal within the first 5-7 days. The remaining transfected hepatocytes persisted for 2-4 weeks and divided one or two times without forming a clone. After 10 attempts of transfection human fetal hepatocytes using the same transfection system, we obtained one stable human fetal hepatocytes cell line which was able albumin secretion urea production and glucose consumption. We established a conditional human fetal hepatocytes cell line with mesenchymal characteristics. Thus immortalization of human fetal hepatocytes cell line by telomerase biology offers a great challenge to examine basic biological mechanisms which are directly related to human and best cell source having unlimited population doubling for bioartificial support without any risk of replicative senescence and pathogenic risks.

Revival, characterization, and hepatitis B virus infection of cryopreserved human fetal hepatocytes

Primary human hepatocytes are considered the ideal cellular model for in-vitro studies of liver-specific pathology, such as hepatitis B virus (HBV) infection. However, poor accessibility, limited cell numbers, and lot-to-lot variation of primary human hepatocytes limit their broad application. Human fetal hepatocytes were isolated from postmortem embryonic liver tissues by two-step collagenase perfusion and cryopreserved. A monolayer of cryopreserved human fetal hepatocytes was established by optimizing such conditions as cell density and viability and purification of viable cells by Percoll. Finally, revived human fetal hepatocytes were characterized and infected with HBV. A large number of viable human fetal hepatocytes could be isolated and cryopreserved, with seeding density and viability being critical for the establishment of a compact monolayer culture. Using low-viability cryopreserved human fetal hepatocytes, a typical monolayer was established by purification with Percoll. The revived cells were actively proliferative, showed identical morphologic characteristics to non-cryopreserved cells, and had a typical hepatic gene expression profile. Moreover, this optimized model was susceptible to HBV infection and could be used to screen entry inhibitors against HBV infection. In conclusion, these methods can be used on human fetal hepatocytes to provide a cell bank for studies of the early stages of HBV infection.

Shortcut Route for Generation of Functional Hepatocyte Cells from Human Skin Allogenically for Autologous Treatment of Chronic Liver Diseases

Zhu Sa, Rezvani Mb, Harbell Jc, Mattis ANb,d,e, Wolfe ARf, Benet LZf, Willenbring Hb,c,d, Ding Sa,g. Mouse liver repopulation with hepatocytes generated from human fibroblasts. Nature. 2014;508(7494):93–97.

Retroviral insertional mutagenesis in telomerase-immortalized hepatocytes identifies RIPK4 as novel tumor suppressor in human hepatocarcinogenesis

Carcinogenesis is a multistep process involving alterations in various cellular pathways. The critical genetic events driving the evolution of primary liver cancer, specifically hepatoblastoma and hepatocellular carcinoma (HCC), are still poorly understood. However, telomere stabilization is acknowledged as prerequisite for cancer progression in humans. In this project, human fetal hepatocytes were utilized as a cell culture model for untransformed, proliferating human liver cells, with telomerase activation as first oncogenic hit. To elucidate critical downstream genetic events driving further transformation of immortalized liver cells, we used retroviral insertional mutagenesis as an unbiased approach to induce genetic alterations. Following isolation of hyperproliferating, provirus-bearing cell clones, we monitored cancer-associated growth properties and characterized changes toward a malignant phenotype. Three transformed clones with the ability to form colonies in soft agar were expanded. As proof-of-principle for our experimental setup, we identified a transforming insertion on chromosome 8 within the pleiomorphic adenoma gene 1 (PLAG1), resulting in a 20-fold increase in PLAG1 expression. Upregulation of PLAG1 has already been described to promote human hepatoblastoma development. In a separate clone, a transforming insertion was detected in close proximity to the receptor-interacting serine-threonine kinase 4 (RIPK4) with an approximately eightfold suppression in RIPK4 expression. As validation for this currently unknown driver in hepatocarcinogenesis, we examined RIPK4 expression in human HCC samples and confirmed a significant suppression of RIPK4 in 80% of the samples. Furthermore, overexpression of RIPK4 in transformed human fetal hepatocytes resulted in an almost complete elimination of anchorage-independent growth. On the basis of these data, we propose RIPK4 as a novel putative tumor suppressor in human hepatocarcinogenesis.