Primary Human Fetal Hepatocytes Research Articles

Gestational diabetes and maternal obesity are associated with sex-specific changes in miRNA and target gene expression in the fetus.

Background/ObjectivePregnancies complicated by gestational diabetes (GDM) or maternal obesity have been linked to the development of diabetes, obesity and fatty liver disease later in life with sex-specific manifestations. Alterations in miRNA expression in offspring exposed to GDM and maternal obesity and effects on hepatic development are unknown. Here we describe how exposure to maternal obesity in utero leads to sex-specific changes in miRNA and target gene expression in human fetal liver.MethodsCandidate miRNA expression was measured in 2nd trimester amniotic fluid (AF) from women with GDM. Targets of differentially expressed miRNAs were determined and pathway enrichment of target genes was performed. MiRNA and target gene expression were measured in a separate cohort of 2nd trimester primary human fetal hepatocytes (PHFH) exposed to maternal obesity via QPCR and western blot. All studies were IRB approved.ResultsGDM exposed AF had significant increases in miRNAs 199a-3p, 503-5p, and 1268a (fold change (FC) ≥1.5, p<0.05). Female offspring specific analysis showed enrichment in miRNAs 378a-3p, 885-5p, and 7-1-3p (p<0.05). MiRNA gene targets were enriched in hepatic pathways. Key genes regulating de novo lipogenesis were upregulated in obesity exposed PHFH, especially in males. Significantly altered miRNAs in GDM AF were measured in obese exposed PHFH, with consistent increases in miRNAs 885-5p, 199-3p, 503-5p, 1268a and 7-1-3p (FC ≥1.5, p<0.05). Female PHFH exposed to maternal obesity had increased expression of miR-885-5p, miR-199-3p, miR-503-5p, miR-1268s and miR-7-1-3p, (p<0.05), corresponding to decreased target genes expression for ABCA1, PAK4 and INSR. In male PHFHs, no miRNA changes were measured but there was increased expression of ABCA1, PAK4, and INSR (p<0.05).ConclusionOur data suggest sex-specific changes in miRNA and gene expression in PHFH may be one mechanism contributing to the sexual dimorphism of metabolic disease in offspring exposed to GDM and maternal obesity in utero.

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&amp;lt;0.05) and sex specific analysis showed enrichment in miRNAs 378a-3p, 885-5p, and 7-1-3p in female offspring samples (p&amp;lt;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&amp;lt;0.05). Female PHFH exposed to MOB were enriched for miRNAs 885-5p, 199-3p, 503-5p, and 7-1-3p (p&amp;lt;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&amp;lt;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.

Effect of fibronectin on HBV infection in primary human fetal hepatocytes in vitro

This study was carried out to investigate the effect of fibronectin (FN) on primary cultured human fetal hepatocytes infected by hepatitisB virus (HBV) invitro. Human fetal hepatocytes infected by HBV were cultured on culture dishes coated with FN invitro. HBsAg and HBeAg in supernatants were detected by ELISA, HBcAg in nuclei by immunohistochemistry, HBV DNA by fluorescent quantitative PCR and covalently closed circular DNA (cccDNA) by nested PCR. The results were compared with cells cultured on dishes that were not coated with FN. Positivity for HBsAg and HBeAg in supernatants appeared from day5 on the non-coated dishes, while positivity for HBsAg and HBeAg appeared from day1 on the coated dishes. Positivity for HBcAg in the nuclei on the non‑coated dishes was displayed from day2 with a positive rate of 15%, while positivity for HBcAg on the coated dishes was displayed from day1 with a positive rate of >90%. HBV DNA in cells on the non-coated dishes was detected from day4, while that on the coated dishes was detected from day1. The non-coated dishes were positive for cccDNA from day8, while the coated dishes were positive from day2. FN coating is capable of accelerating HBV infection in primary cultured fetal hepatocytes invitro, increasing the duration of HBV infection and the number of infected cells.

Transplantation of Immortalized Human Fetal Hepatocytes Prevents Acute Liver Failure in 90% Hepatectomized Mice

Aim The aim of this study was to investigate whether human fetal hepatocytes are amenable to simian virus 40 large T-antigen (SV40Tag) mediated immortalization and whether the immortalized cells rescue mice with acute liver failure induced by 90% hepatectomy. Methods We constructed a retroviral vector expressing a thermolabile mutant SV40Tag for transfer into primary human fetal hepatocytes. We quantitatively detected the synthetic ability for albumin and urea by the immortalized cells, which were subcutaneously inoculated into mice with severe combined immunodeficiency (SCID) to evaluate tumorigenzcity. The immortalized cells were also transplanted into the spleens of mice with acute liver failure. Results One clone resulting after selection, referred to as HepCL, was highly differentiated, growing steadily in a chemically defined serum-free medium. HepCL cells were positive for albumin, cytokeratin 18, and cytokeratin 19 immunocytochemical staining. The average synthetic efficacies of HepCL cells for albumin and urea were comparable to that of unmodified primary human fetal hepatocytes. The population doubling time of HepCL cells in the logarithmic growth phase was approximately 17 hours. HepCL cells showed no oncogenicity in immunodeficient mice at 16 months. Mice receiving HepCL cells (G1) and primary human fetal hepatocytes (G2) showed significantly lower blood ammonia levels after 90% hepatectomy. Pairwise comparisons between the 4 groups showed that xenotransplantation of HepCL (G1) or primary fetal hepatocytes (G2) significantly improved survivals of recipient mice. Conclusions HepCL may be useful as a source of hepatic function for cell-based therapeutics in acute liver failure.

Advanced Glycation End Products Upregulate C‐reactive Protein Synthesis by Human Hepatocytes Through Stimulation of Monocyte IL‐6 and IL‐1β Production

Patients with chronic renal failure are characterized by increased plasma levels of C-reactive protein (CRP) and advanced glycation end products (AGE). AGE have been identified as a class of proinflammator mediators. To investigate whether AGE can stimulate hepatocytes to produce CRP, primary human fetal hepatocytes (HFH) were incubated with AGE-modified human serum albumin (AGE-HSA) or conditioned medium from AGE-HSA-stimulated monocytes (AGE-MCM). CRP concentrations in the supernatants were determined by an ELISA and CRP mRNA levels were determined by a quantitative RT-PCR. Exposure of HFH with AGE-HSA for 12-72 h did not change CRP concentrations in the supernatants. CRP protein and mRNA expression were significantly upregulated in a time- and dose-dependent manner when HFH were incubated with AGE-MCM. This stimulating effect was partially inhibited when AGE-MCM were preincubated with antibodies against interleukin-6 (anti-IL-6), interleukin-1 beta (anti-IL-1 beta), or soluble IL-1 receptor and was completely inhibited when AGE-MCM were preincubated with anti-IL-6 and anti-IL-1 beta simultaneously. The inhibiting effect did not occur when AGE-MCM was preincubated with antibody of tumour necrosis factor-alpha (anti-TNF-alpha) and soluble TNF receptor. Exposure of HFH with exogenous IL-6 and IL-1 beta, at the same concentrations as contained in AGE-MCM, also increased CRP production, but exogenous TNF-alpha had no effect. These results suggest that AGE cannot directly stimulate hepatocytes to produce CRP, but rather indirectly enhance CRP expression via stimulation of IL-6 and IL-1 beta production by human monocytes.

A Dataset of Human Liver Proteins Identified by Protein Profiling Via Isotope-coded Affinity Tag (ICAT) and Tandem Mass Spectrometry

Proteins from human liver carcinoma Huh7 cells, representing transformed liver cells, and cultured primary human fetal hepatocytes (HFH) and human HH4 hepatocytes, representing nontransformed liver cells, were extracted and processed for proteome analysis. Proteins from stimulated cells (interferon-alpha treatment for the Huh7 and HFH cells and induction of hepatitis C virus [HCV] proteins for the HH4 cells) and corresponding control cells were labeled with light and heavy cleavable ICAT reagents, respectively. The labeled samples were combined, trypsinized, and subject to cation-exchange and avidin-affinity chromatographies. The resulting cysteine-containing peptides were analyzed by microcapillary LC-MS/MS. The MS/MS spectra were initially analyzed by searching the human International Protein Index database using the SEQUEST software (1). Subsequently, new statistical algorithms were applied to the collective SEQUEST search results of each experiment. First, the PeptideProphet software (2) was applied to discriminate true assignments of MS/MS spectra to peptide sequences from false assignments, to assign a probability value for each identified peptide, and to compute the sensitivity and error rate for the assignment of spectra to sequences in each experiment. Second, the ProteinProphet software (3) was used to infer the protein identifications and to compute probabilities that a protein had been correctly identified, based on the available peptide sequence evidence. The resulting protein lists were filtered by a ProteinProphet probability score p > or = 0.5, which corresponded to an error rate of less than 5%. A total of 1,296, 1,430, and 1,476 proteins or related protein groups were identified in three subdatasets from the Huh7, HFH, and HH4 cells, respectively. In total, these subdatasets contained 2,486 unique protein identifications from human liver cells. An increase of the threshold to p > or = 0.9 (corresponding to an error rate of less than 1%) resulted in 2,159 unique protein identifications (1,146, 1,235, and 1,318 for the Huh7, HFH, and HH4 cells, respectively).

Spheroid formation and functional restoration of human fetal hepatocytes on poly–amino acid-coated dishes after serial proliferation

Primary human fetal hepatocytes proliferated in monolayer culture up to the 9th passage. During proliferation, the cells changed their morphology from a fibroblast-like shape after inoculation to an epithelia-like polygonal shape after they reached confluence. The proliferation was associated with the loss of ammonia detoxification capacity, which is essential for the function of bioartificial liver. The cells formed spheroids on a poly-glutamic acid- or poly-aspartic acid-coated polystyrene dish that had a negatively charged surface at neutral pH. However, the cells did not form spheroids on a poly-lysine- or poly-arginine-coated dish that had a positively charged surface, which is reportedly suitable to form spheroids for adult hepatocytes. The activity of cytochrome P450 (CYP 1A1, CYP1A2) of the cells in spheroid culture was about twice as high as that of the cells in monolayer culture. The ammonia detoxification activity of the cells was restored in spheroid culture by treatment with 2% dimethylsulfoxide. These results suggest that the conditions for human fetal hepatocytes to form spheroids are different from that for adult hepatocytes, and the use of poly-glutamic acid or poly-aspartic acid coating may improve spheroid culture of proliferative human fetal hepatocytes.

Treatment of surgically induced acute liver failure with transplantation of highly differentiated immortalized human hepatocytes.

Primary human hepatocytes are an ideal source of hepatic function in bioartficial liver (BAL), but the shortage of human livers available for hepatocyte isolation limits this modality. To resolve this issue, primary human fetal hepatocytes were immortalized using simian virus 40 large T antigen. One of the immortal cell lines, OUMS-29, showed highly differentiated liver functions. Intrasplenic transplantation of OUMS-29 cells protected 90% hepatectomized rats from hyperammonemia and significantly prolonged their survival. Essentially unlimited availability of OUMS-29 cells supports their clinical use for BAL treatment.

Transplantation of highly differentiated immortalized human hepatocytes to treat acute liver failure.

Temporary support of a damaged liver by a bioartificial liver (BAL) devise is a promising approach for the treatment of acute liver failure. Although human primary hepatocytes are an ideal source of hepatic function in BAL, shortage of human livers available for hepatocyte isolation is the limiting factor for the use of this modality. A clonal human hepatocyte cell line that can grow economically in culture and exhibit liver-specific functions should be an attractive solution to this problem. To test this alternative, primary human fetal hepatocytes were immortalized using Simian virus 40 large T antigen. To investigate the potential of the immortalized cells for BAL, we transplanted the cells into the spleen of adult rats and performed a 90% hepatectomy 12 hr later. One of the cloned human liver cell lines, OUMS-29, showed highly differentiated liver functions. Intrasplenic transplanting of 20x10(6) OUMS-29 cells protected the animals from hyperammonemia and the associated hepatic encephalopathy. Survival was significantly prolonged in 90% of hepatectomized rats receiving OUMS-29 cells. A highly differentiated immortalized human hepatocyte cell line, OUMS-29, was able to provide metabolic support during acute liver failure induced by 90% hepatectomy in rats. Essentially unlimited availability of OUMS-29 cells may be clinically useful for BAL treatment.

TRANSPLANTATION OF HIGHLY DIFFERENTIATED IMMORTALIZED HUMAN HEPATOCYTES TO TREAT ACUTE LIVER FAILURE

26 Introduction: Because the liver has an enormous capacity for regeneration, temporary support of a damaged liver by a bioartficial liver (BAL) devise is a promising approach for the treatment of acute liver failure. Large quantities of viable and actively functioning hepatocytes are required for BAL treatment of ALF or as a bridge to liver transplantation. However, the shortage of human livers available for hepatocyte isolation severely limits the use of this modality. A clonal hepatocyte cell line that could be grown economically in tissue culture and would exhibit liver-specific function would be an attractive solution to this problem. Purpose: To test this alternative, primary human fetal hepatocytes were immortalized by transfection with a plasmid pSV3neo containing the genes encoding Simian virus 40 large T antigen (SV40Tag) and neomycin phosphotransferase. One of the immortalized cell lines, OUMS-29 secreted albumin, expressed mRNAs for CYP 1A1, 1A2 and 2E1 and showed an ammonia removal activity. OUMS-29 cells were not tumorigenic after transplantation into athymic nude mice. In order to investigate the potential of OUMS-29 cells, cells were transplanted into the spleen of rats followed by 90% hepatectomy. Methods: Rats were divided into the following groups: Group1 (G1:n=12), intrasplenic transplantation of 20×106 OUMS-29 cells; Group2 (G2:n=5), intrasplenic transplantation of 20×106 HepG2 cells (transformed human liver cells); Group3 (G3:n=10), intrasplenic injection of 0.5ml medium. All rats underwent 90% hepatectomy 12 hr after transplantation. Results: Rats in G1 showed a significantly lower increase in blood ammonia levels after 90% hepatectomy. Six out of 12 animals in G1 survived more than 48 hr after hepatectomy, whereas all animals in G2 and G3 died of acute liver failure within 28 hr. Survival of G1 rats was significantly prolonged compared to G1 and G2 rats. Conclusions: Intrasplenic transplantation of OUMS-29 cells protected animals from hyperammonemia and prolonged survival in 90% hepatectomized rats. The present studies show that primary human hepatocytes can be immortalized, expanded in culture with retaining a highly differentiated liver function, and are capable of providing metabolic support in acute liver insufficiency. OUMS-29 cells offer the advantages of unlimited availability, uniformity, sterility and freedom from infectious pathogens and these cells might be clinically used for hepatic function in BAL to improve the lives of a large number of patients with ALF.

Production and Effect of Infectious Dane Particles in Transgenic Mice

We have demonstrated by immunoelectron microscopy that 42‐nm particles with double‐shelled structures characteristic of Dane particles are present in the serum of transgenic mice, 1.2HB‐BS 10, carrying partly duplicated hepatitis B virus (HBV) genome. Furthermore, these particles were shown to infect primary human fetal hepatocytes as demonstrated by the elevation of MBV surface antigen (HBsAg) in the culture medium. HBV DNA is known to be expressed in a liver‐ and kidney‐specific manner in the adult mouse, so we examined the developmental expression of viral antigens. In the liver, viral antigens (HBsAg and HBV e antigen) began to be expressed before birth and the level of expression showed a sharp rise after birth. On the other hand, in the kidney, viral antigens began to be expressed after birth. Serum levels of viral antigens were roughly proportional to the levels of expression in the liver, suggesting that the liver is the main source for viral antigens in the serum. None of these transgenic mice produced anti‐HBs or anti‐HBV core response or showed biochemical or pathological change up to at least 24 months of age. All these results suggest that infectious viral particles can be produced in transgenic mice, and that expression and replication of HBV DNA are not toxic in vivo.

An in vitro system for infection with hepatitis B virus that uses primary human fetal hepatocytes.

An in vitro culture of human fetal hepatocytes has been employed for infection by hepatitis B virus (HBV) virions that are produced by an established human hepatoma cell line, HB 611. HBV surface antigen and e antigen were released into the medium 3-4 days after infection, and production continued thereafter. RNA synthesis with similar kinetics was observed. Viral DNA replication started 2 days after infection, and replicative HBV DNA that included relaxed circles, single-stranded minus strands, and closed circles accumulated during 16 days of incubation. Immunofluorescent study using fluorescein isothiocyanate-labeled rabbit antisera directed against HBV core antigen revealed that this antigen is present in the nuclei in 12% of the infected cells. Particles containing HBV DNA were detected in the culture medium and were infectious. Thus, this in vitro infection system closely mimics infection in vivo and it allows detailed studies on early events associated with human HBV entry into cells and subsequent replication and integration.