Adult Human Liver Research Articles

Single base resolution analysis of 5-hydroxymethylcytosine in 188 human genes: implications for hepatic gene expression.

To improve the epigenomic analysis of tissues rich in 5-hydroxymethylcytosine (hmC), we developed a novel protocol called TAB-Methyl-SEQ, which allows for single base resolution profiling of both hmC and 5-methylcytosine by targeted next-generation sequencing. TAB-Methyl-SEQ data were extensively validated by a set of five methodologically different protocols. Importantly, these extensive cross-comparisons revealed that protocols based on Tet1-assisted bisulfite conversion provided more precise hmC values than TrueMethyl-based methods. A total of 109 454 CpG sites were analyzed by TAB-Methyl-SEQ for mC and hmC in 188 genes from 20 different adult human livers. We describe three types of variability of hepatic hmC profiles: (i) sample-specific variability at 40.8% of CpG sites analyzed, where the local hmC values correlate to the global hmC content of livers (measured by LC-MS), (ii) gene-specific variability, where hmC levels in the coding regions positively correlate to expression of the respective gene and (iii) site-specific variability, where prominent hmC peaks span only 1 to 3 neighboring CpG sites. Our data suggest that both the gene- and site-specific components of hmC variability might contribute to the epigenetic control of hepatic genes. The protocol described here should be useful for targeted DNA analysis in a variety of applications.

Transcription Factors and Medium Suitable for Initiating the Differentiation of Human-Induced Pluripotent Stem Cells to the Hepatocyte Lineage.

Transcription factors and culture media were investigated to determine the condition to initiate the differentiation of human-induced pluripotent stem (iPS) cells most efficiently. The expression of genes in human adult liver was compared with that in 201B7 cells (iPS cells) using cDNA microarray analysis. Episomal plasmids expressing transcription factors were constructed. 201B7 cells were transfected with the episomal plasmids and cultured in ReproFF (feeder-free media maintaining pluripotency), Leibovitz-15 (L15), William's E (WE), or Dulbecco's modified Eagle medium/Nutrient F-12 Ham (DF12) for 7 days. RNA was isolated and subjected to real-time quantitative PCR to analyze the expression of alpha-feto protein (AFP) and albumin. cDNA microarray analysis revealed 16 transcription factors that were upregulated in human adult liver relative to that in 201B7 cells. Episomal plasmids expressing these 16 genes were transfected into 201B7 cells. CCAAT/enhancer-binding protein alpha (CEBPA), CCAAT/enhancer-binding protein beta (CEBPB), forkhead box A1 (FOXA1), and forkhead box A3 (FOXA3) up-regulated AFP and down-regulated Nanog. These four genes were further analyzed. The expression of AFP and albumin was the highest in 201B7 cells transfected with the combination of CEBPA, CEBPB, FOXA1, and FOXA3 and cultured in WE. The combination of CEBPA, CEBPB, FOXA1, and FOXA3 was suitable for 201B7 cells to initiate differentiation to the hepatocyte lineage and WE was the most suitable medium for culture after transfection. J. Cell. Biochem. 117: 2001-2009, 2016. © 2016 Wiley Periodicals, Inc.

Anti-hepatitis C virus potency of a new autophagy inhibitor using human liver slices model.

To evaluate the antiviral potency of a new anti-hepatitis C virus (HCV) antiviral agent targeting the cellular autophagy machinery. Non-infected liver slices, obtained from human liver resection and cut in 350 μm-thick slices (2.7 × 10(6) cells per slice) were infected with cell culture-grown HCV Con1b/C3 supernatant (multiplicity of infection = 0.1) cultivated for up to ten days. HCV infected slices were treated at day 4 post-infection with GNS-396 for 6 d at different concentrations. HCV replication was evaluated by strand-specific real-time quantitative reverse transcription - polymerase chain reaction. The infectivity titers of supernatants were evaluated by foci formation upon inoculation into naive Huh-7.5.1 cells. The cytotoxic effect of the drugs was evaluated by lactate dehydrogenase leakage assays. The antiviral efficacy of a new antiviral drug, GNS-396, an autophagy inhibitor, on HCV infection of adult human liver slices was evidenced in a dose-dependent manner. At day 6 post-treatment, GNS-396 EC50 was 158 nmol/L without cytotoxic effect (compared to hydroxychloroquine EC50 = 1.17 μmol/L). Our results demonstrated that our ex vivo model is efficient for evaluation the potency of autophagy inhibitors, in particular a new quinoline derivative GNS-396 as antiviral could inhibit HCV infection in a dose-dependent manner without cytotoxic effect.

Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells

Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications.

Hepatocytic Differentiation Potential of Human Fetal Liver Mesenchymal Stem Cells: In Vitro and In Vivo Evaluation.

In line with the search of effective stem cell population that would progress liver cell therapy and because the rate and differentiation potential of mesenchymal stem cells (MSC) decreases with age, the current study investigates the hepatogenic differentiation potential of human fetal liver MSCs (FL-MSCs). After isolation from 11-12 gestational weeks' human fetal livers, FL-MSCs were shown to express characteristic markers such as CD73, CD90, and CD146 and to display adipocytic and osteoblastic differentiation potential. Thereafter, we explored their hepatocytic differentiation potential using the hepatogenic protocol applied for adult human liver mesenchymal cells. FL-MSCs differentiated in this way displayed significant features of hepatocyte-like cells as demonstrated in vitro by the upregulated expression of specific hepatocytic markers and the induction of metabolic functions including CYP3A4 activity, indocyanine green uptake/release, and glucose 6-phosphatase activity. Following transplantation, naive and differentiated FL-MSC were engrafted into the hepatic parenchyma of newborn immunodeficient mice and differentiated in situ. Hence, FL-MSCs appeared to be interesting candidates to investigate the liver development at the mesenchymal compartment level. Standardization of their isolation, expansion, and differentiation may also support their use for liver cell-based therapy development.

Patterns of gene expression and DNA methylation in human fetal and adult liver.

BackgroundDNA methylation is an important epigenetic control mechanism that has been shown to be associated with gene silencing through the course of development, maturation and aging. However, only limited data are available regarding the relationship between methylation and gene expression in human development.ResultsWe analyzed the methylome and transcriptome of three human fetal liver samples (gestational age 20–22 weeks) and three adult human liver samples. Genes whose expression differed between fetal and adult numbered 7,673. Adult overexpression was associated with metabolic pathways and, in particular, cytochrome P450 enzymes while fetal overexpression reflected enrichment for DNA replication and repair. Analysis for DNA methylation using the Illumina Infinium 450 K HumanMethylation BeadChip showed that 42 % of the quality filtered 426,154 methylation sites differed significantly between adult and fetal tissue (q ≤ 0.05). Differences were small; 69 % of the significant sites differed in their mean methylation beta value by ≤0.2. There was a trend among all sites toward higher methylation in the adult samples with the most frequent difference in beta being 0.1. Characterization of the relationship between methylation and expression revealed a clear difference between fetus and adult. Methylation of genes overexpressed in fetal liver showed the same pattern as seen for genes that were similarly expressed in fetal and adult liver. In contrast, adult overexpressed genes showed fetal hypermethylation that differed from the similarly expressed genes. An examination of gene region-specific methylation showed that sites proximal to the transcription start site or within the first exon with a significant fetal-adult difference in beta (>0.2) showed an inverse relationship with gene expression.ConclusionsNearly half of the CpGs in human liver show a significant difference in methylation comparing fetal and adult samples. Sites proximal to the transcription start site or within the first exon that show a transition from hypermethylation in the fetus to hypomethylation or intermediate methylation in the adult are associated with inverse changes in gene expression. In contrast, increases in methylation going from fetal to adult are not associated with fetal-to-adult decreased expression. These findings indicate fundamentally different roles for and/or regulation of DNA methylation in human fetal and adult liver.

BCRP protein levels do not differ regionally in adult human livers, but decline in the elderly

The aim of this study was to characterize the ontogeny and variability of the BCRP (ABCG2) transporter in healthy human liver. Levels of BCRP mRNA and protein were determined with q-RT-PCR and western blot in a cohort of 87 human livers aged from 7 days to 87 years. A study of the regional expression of BCRP within adult livers was also performed in a nested cohort of 14 individuals with multiple samples per person collected from pre-selected sites. Levels of BCRP mRNA were not significantly different at any age, but protein levels for BCRP were lower in the elderly compared with adults (p < 0.001) and children (p < 0.05). The intra-liver levels of BCRP protein ranged approximately 6.5-fold and inter-liver BCRP protein varied 8.5-fold in the cohort. No differences in BCRP mRNA or protein were observed with sex or ethnicity, although higher levels of BCRP mRNA were observed in livers from overweight individuals (Body Mass Index ≥ 25–29.9) as compared to underweight or ideal weight individuals. There were no differences in the levels of BCRP mRNA or protein in different regions of the large lobe (n = 3 regions), small lobe (n = 3 regions), directly adjacent to the portal vein or directly adjacent to the common bile duct. This indicates that BCRP researchers can source tissue from all parts of the adult liver without artificial bias in their results. Lower BCRP protein expression in the elderly may be associated with compromised xeno- and endobiotic transport.

Anatomical variations in the pattern of the right hepatic veins draining the posterior segment of the right lobe of the liver

Background: The pattern of drainage in the right posterior lobe of liver varies considerably. The knowledge of this variation is very important while performing various surgeries on the right posterior lobe. Aim: A study was conducted to see the variations in the pattern of drainage of posterior segment of the right lobe of liver. The aim was to see the variations of right hepatic vein and small accessory hepatic veins draining the posterior segment, the presence of which led to modifications in drainage of posterior segment. Material and Methods: Sixty formalin fixed adult human liver specimens were dissected manually. Results: According to the pattern of drainage of tributaries of right hepatic vein, the right hepatic vein was classified into type I, type II, type III and type IV. According to presence of inferior right hepatic vein, three types of drainage of posterior lobe were seen: Type I, (76.36%) right hepatic vein was large, draining wide area of posterior segment. A small inferior right hepatic vein drained the small area of posterior segment. In Type II, (19.92%) both right hepatic and inferior right hepatic veins were medium sized draining the posteroinferior segment of the right lobe concomitantly. In Type III, (32%) accessory veins, the middle right hepatic vein drained the posterosuperior (VII) as well as the posteroinferior (VI) segment. In one specimen, there were numerous middle right hepatic veins draining the right posterior segment. The knowledge of anatomic relationship of veins draining right lobe, is important in performing right posterior segmentectomy. Conclusion: For safe resection of the liver, the complex anatomy of the distribution of the tributaries of the right hepatic vein and the accessory veins have to be studied prior to any surgery done on liver.

P223: Human adult liver stem cells from patient samples

P223: Human adult liver stem cells from patient samples

Human adult liver stem cells generated from patient samples

Basic and translational research on primary human hepatocytes is currently hindered by the limited availability of functional human hepatocytes. Moreover, once isolated from tissue samples, they rapidly loose their function and do not proliferate when cultured in vitro. This problem could be overcome by using human liver stem cells, propagated in vitro and subsequently differentiated into cells of the different liver cell types, ideally reaching full maturation comparable to native cells in vivo.

Anatomical variations in the pattern of the right hepatic veins draining the posterior segment of the right lobe of the liver.

The pattern of drainage in the right posterior lobe of liver varies considerably. The knowledge of this variation is very important while performing various surgeries on the right posterior lobe. A study was conducted to see the variations in the pattern of drainage of posterior segment of the right lobe of liver. The aim was to see the variations of right hepatic vein and small accessory hepatic veins draining the posterior segment, the presence of which led to modifications in drainage of posterior segment. Sixty formalin fixed adult human liver specimens were dissected manually. According to the pattern of drainage of tributaries of right hepatic vein, the right hepatic vein was classified into type I, type II, type III and type IV. According to presence of inferior right hepatic vein, three types of drainage of posterior lobe were seen: Type I, (76.36%) right hepatic vein was large, draining wide area of posterior segment. A small inferior right hepatic vein drained the small area of posterior segment. In Type II, (19.92%) both right hepatic and inferior right hepatic veins were medium sized draining the posteroinferior segment of the right lobe concomitantly. In Type III, (32%) accessory veins, the middle right hepatic vein drained the posterosuperior (VII) as well as the posteroinferior (VI) segment. In one specimen, there were numerous middle right hepatic veins draining the right posterior segment. The knowledge of anatomic relationship of veins draining right lobe, is important in performing right posterior segmentectomy. For safe resection of the liver, the complex anatomy of the distribution of the tributaries of the right hepatic vein and the accessory veins have to be studied prior to any surgery done on liver.

Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver

SummaryDespite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy.

Regulation of flavin-containing mono-oxygenase (Fmo3) gene expression by steroids in mice and humans.

Abstract Flavin-containing mono-oxygenases (FMOs) are a family of microsomal chemical- and drug-metabolizing enzymes. FMO3 is a major FMO form in adult mouse and human liver. FMO3 mutations have been associated with the incidence and severity of trimethylaminuria (TMAU), a metabolic disorder characterized by the inability of the affected individual to metabolize the odorous trimethylamine to its non-odorous N-oxide. In addition to this primary genetic form, there are other forms of TMAU that support the hypothesis that FMO3 activity may be modulated by steroid hormones. To understand the molecular mechanism involved in the regulation of Fmo3 gene expression by steroid hormones, we performed this study in an in vitro cellular system, mouse liver cells, and on the human FMO3 gene. Dexamethasone, 5α-dihydrotestosterone, thyroid hormone, and progesterone had no effect on the accumulation of Fmo3 mRNA. The use of increased concentration of theophylline inhibited estrogen receptor α (ERα)-mediated transcription of Fmo3 mRNA. 17β-Estradiol inhibited Fmo3 mRNA accumulation. The use of ICI 164,384 abolished the inhibitory effect induced by estrogen. Gel-shift analyses showed a binding in the 5' region of the Fmo3 gene. This binding was abrogated by an excess of a cDNA containing an estrogen-responsive element. An estrogen-binding site was also present in the first intron of the human gene, as demonstrated by the gel-shift assay. Supershift experiments confirmed the binding of ERα in both mouse and human samples. Furthermore, chromatin immunoprecipitation assay confirmed the binding of ERα in the promoter region of mouse Fmo3 and in the first intron of the human FMO3 gene. Thus, 17β-estradiol plays a fundamental role in the regulation of Fmo3 gene transcription.

Human liver stem cells and derived extracellular vesicles improve recovery in a murine model of acute kidney injury.

IntroductionSeveral cellular sources of stem cells have been tested in the attempt to yield innovative interventions in acute kidney injury (AKI). Human liver stem cells (HLSCs) are cells isolated from the normal adult human liver which are gaining attention for their therapeutic potential. In the present study, we investigated whether HLSCs and the derived extracellular vesicles may promote tubular regeneration after AKI induced by glycerol injection in severe-combined immune-deficient mice.MethodsHLSCs were expanded and conditioned medium (CM) and extracellular vesicles (EVs) were purified. HLSCs and their bioproducts were tested in a model of AKI induced by intra-muscle glycerol injection. Renal function and morphology were evaluated five days after induction of damage. The effect of EVs on proliferation and apoptosis of murine renal tubular cells was tested in vitro.ResultsWe found that intravenous injection of 3.5×105 HLSCs into mice three days after induction of AKI significantly improved functional and morphological recovery. The injection of HLSCs decreased creatinine and urea, as well as hyaline cast formation, tubular necrosis and enhanced in vivo tubular cell proliferation. The effect of soluble factors release by HLSCs in the regenerative processes was also studied. CM produced by HLSCs, mimicked the effect of the cells. However, depletion of EVs significantly reduced the functional and morphological recovery of CM. Moreover, we found that purified HLSC-derived EVs ameliorated renal function and morphology in a manner comparable to the cells. In vitro HLSC-derived EVs were shown to stimulate proliferation and inhibit apoptosis of murine renal tubular cells.ConclusionsThese results indicate that HLSCs increase recovery after AKI. EVs are the main component of HLSC-derived CM capable of promoting regeneration in experimental AKI.

Expression and distribution of immunoglobulin G in the normal liver, hepatocarcinoma and postpartial hepatectomy liver

The liver has the extraordinary properties of regeneration and immune tolerance; however, the mechanisms governing these abilities are poorly understood. To address these questions, we examined the possible expression of immunoglobulins in the human and rat liver and the relationship of IgG expression to hepatocyte proliferation, metastasis, apoptosis and immune tolerance. Immunohistochemistry, in situ hybridization, laser-guided microdissection and reverse transcription-PCR were performed to examine the expression of IgG in normal human and rat liver, severe combined immunodeficient mouse (SCID) liver and human liver cancers and corresponding cell lines. Small interfering RNA (siRNA) was transfected into cultured hepatocarcinoma cells to downregulate the expression of IgG heavy chain genes. Cell proliferation and apoptosis were assayed with flow cytometry. Cell metastasis was assayed with a Transwell cell assay. Partial hepatectomy (70%) was performed in rats to examine the relationship between hepatocyte IgG and hepatocyte proliferation. IgG, together with essential enzymes for its synthesis, were expressed in the cytoplasm of hepatocytes of normal adult human and hepatoma patients and rat livers, SCID mouse liver and BRL-3A, L-02 and HepG-2 cell lines. Downregulation of IgG inhibited cell proliferation and metastasis and promoted apoptosis. Postsurgery livers expressed significantly more IgG than the livers before surgery and decreased to the original levels when hepatocytes stopped regeneration. IgA and IgM but not IgD and IgE were also positive in hepatocytes. Our findings demonstrate that normal and malignant hepatocytes are capable of synthesizing immunoglobulin, which has important roles in hepatocyte proliferation, apoptosis and cancer growth with profound clinical implications.

Ligand-activated PPARα-dependent DNA demethylation regulates the fatty acid β-oxidation genes in the postnatal liver.

The metabolic function of the liver changes sequentially during early life in mammals to adapt to the marked changes in nutritional environment. Accordingly, hepatic fatty acid β-oxidation is activated after birth to produce energy from breast milk lipids. However, how it is induced during the neonatal period is poorly understood. Here we show DNA demethylation and increased mRNA expression of the fatty acid β-oxidation genes in the postnatal mouse liver. The DNA demethylation does not occur in the fetal mouse liver under the physiologic condition, suggesting that it is specific to the neonatal period. Analysis of mice deficient in the nuclear receptor peroxisome proliferator-activated receptor α (PPARα) and maternal administration of a PPARα ligand during the gestation and lactation periods reveal that the DNA demethylation is PPARα dependent. We also find that DNA methylation of the fatty acid β-oxidation genes are reduced in the adult human liver relative to the fetal liver. This study represents the first demonstration that the ligand-activated PPARα-dependent DNA demethylation regulates the hepatic fatty acid β-oxidation genes during the neonatal period, thereby highlighting the role of a lipid-sensing nuclear receptor in the gene- and life-stage-specific DNA demethylation of a particular metabolic pathway.

Genetic and epigenetic regulation of gene expression in fetal and adult human livers.

BackgroundThe liver plays a central role in the maintenance of homeostasis and health in general. However, there is substantial inter-individual variation in hepatic gene expression, and although numerous genetic factors have been identified, less is known about the epigenetic factors.ResultsBy analyzing the methylomes and transcriptomes of 14 fetal and 181 adult livers, we identified 657 differentially methylated genes with adult-specific expression, these genes were enriched for transcription factor binding sites of HNF1A and HNF4A. We also identified 1,000 genes specific to fetal liver, which were enriched for GATA1, STAT5A, STAT5B and YY1 binding sites. We saw strong liver-specific effects of single nucleotide polymorphisms on both methylation levels (28,447 unique CpG sites (meQTL)) and gene expression levels (526 unique genes (eQTL)), at a false discovery rate (FDR) < 0.05. Of the 526 unique eQTL associated genes, 293 correlated significantly not only with genetic variation but also with methylation levels. The tissue-specificities of these associations were analyzed in muscle, subcutaneous adipose tissue and visceral adipose tissue. We observed that meQTL were more stable between tissues than eQTL and a very strong tissue-specificity for the identified associations between CpG methylation and gene expression.ConclusionsOur analyses generated a comprehensive resource of factors involved in the regulation of hepatic gene expression, and allowed us to estimate the proportion of variation in gene expression that could be attributed to genetic and epigenetic variation, both crucial to understanding differences in drug response and the etiology of liver diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-860) contains supplementary material, which is available to authorized users.

A statistical geometrical description of the human liver for probabilistic occupant models

Realistic numerical assessments of liver injury risk for the entire occupant population require incorporating inter-subject variations into numerical models. Statistical shape models of the abdominal organs have been shown to be useful tools for the investigation of the organ variations and could be applied to the development of statistical computational models. The main objective of this study was to establish a standard procedure to quantify the shape variations of a human liver in a seated posture, and construct three-dimensional (3D) statistical shape boundary models.Statistical shape analysis was applied to construct shape models of 15 adult human livers. Principal component analysis (PCA) was then utilized to obtain the modes of variation, the mean model, and a set of statistical boundary shape models, which were constructed using the q-hyper-ellipsoid approach.The first five modes of a human liver accounted for the major anatomical variations. The modes were highly correlated to the height, thickness, width, and curvature of the liver, and the concavity of the right lobe. The mean model and the principal components were utilized to construct four boundary models of human liver. The statistical boundary model approach presented in this study could be used to develop probabilistic finite element (FE) models. In the future, the probabilistic liver models could be used in FE simulations to better understand the variability in biomechanical responses and abdominal injuries under impact loading.

Transfer of malignant trait to immortalized human cells following exposure to human cancer serum.

BackgroundHuman cancer cells can transfer signaling molecules to neighboring and distant cells predisposing them to malignant transformation. This process might contribute to tumor progression and invasion through delivery of oncogenes or inhibitors of tumor suppressor genes, derived from the primary tumor cells, to susceptible target cells. The oncogenic potential of human cancer serum has been described in immortalized mouse fibroblasts but has not been shown yet in human cells. The objective of this study was to determine whether metastatic cancer patient sera have the ability to induce neoplastic transformation in immortalized human embryonic kidney (HEK293) cells, human embryonic stem cells (hESCs), human mesenchymal stem cells (hMSCs) and human adult liver fibroblasts (hALFs).MethodsEarly passage HEK293 cells, hESCs, hMSCs and hALFs were exposed to cancer patient serum, or cancer cells-derived condition medium for 3 weeks. Treated cells were analyzed for cell proliferation and transformation both in vitro and in vivo.ResultsHEK293 cells exposed to cancer serum increased their proliferative capability and displayed characteristics of transformed cells, as evaluated by in vitro anchorage-independent growth assay and in vivo tumorigenesis in immunodeficient mice. The same phenotypes were acquired when these cells were cultured in cancer cell line conditioned medium suggesting that the putative oncogenic factors present in the serum might derive directly from the primary tumor. Histopathological analyses revealed that the tumors arising from cancer patient serum and conditioned medium-treated HEK293 cells were poorly differentiated and displayed a high proliferative index. In contrast, neither of these phenomena was observed in treated hMSCs and hALFs. Intriguingly enough, hESC-treated cells maintained their self-renewal and differentiation potentials, as shown by in vitro sphere formation assay and in vivo development of teratomas in immunodeficient mice.ConclusionOur results indicate that cancer patients serum is able to induce oncogenic transformation of HEK293 cells and maintain the self-renewal of hESCs. To our knowledge, this is the first study that demonstrates the oncogenic transformation potential of cancer patient serum on human cells. In depth characterization of this process and the molecular pathways involved are needed to confirm its validity and determine its potential use in cancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-014-0086-5) contains supplementary material, which is available to authorized users.

UDP-glucuronosyltransferase 1a enzymes are present and active in the mouse blastocyst.

The UDP-glucuronosyltransferase (UGT) enzymes are critical for regulating nutrients, hormones, and endobiotics, as well as for detoxifying xenobiotics. Human and murine fetuses are known to express glucuronidation enzymes, but there are currently no data prior to implantation. Here we addressed this gap in knowledge and tested whether Ugt enzymes are already present in preimplantation-stage embryos. Blastocysts were obtained after in vitro fertilization with gametes from B6D2F1 hybrid mice and from embryo culture. Protein expression and localization were determined using pan-specific UGT1A and UGT2B, as well as anti-human isoform-specific antibodies. Immunofluorescence analysis showed that blastocysts expressed Ugt1a globally, in the cytoplasm and nuclei of all of the cells. Western blots demonstrated the presence of Ugt1a6 but not Ugt1a1, Ugt1a3, Ugt1a4, or Ugt1a9. The Ugt2b proteins were not detected by either assay. The level of Ugt activity in murine blastocysts was comparable with that of the adult human liver (per milligram of protein), but the activity of β-glucuronidase, an Ugt-partnering enzyme responsible for substrate regeneration, was lower. Altogether, these data confirm that Ugt1a proteins are present and active in preimplantation murine embryos and point to a potential role for these proteins in implantation and early embryonic and fetal development.