Gata4 blocks somatic cell reprogramming by directly repressing Nanog.

Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by ectopic expression of the four factors Oct4, Klf4, Sox2, and Myc. Here, we investigated the role of Gata4 in the reprogramming process and present evidence for a negative role of this family of transcription factors in the induction of pluripotency. Coexpression of Gata4 with Oct4, Klf4, and Sox2 with or without Myc in mouse embryonic fibroblasts greatly impaired reprogramming and endogenous Nanog expression. The lack of Nanog upregulation was associated with a blockade in the transition from the initiation phase of reprogramming to the full pluripotent state characteristic of iPS cells. Addition of Nanog to the reprogramming cocktail blocked the deleterious effects observed with Gata4 expression. Downregulation of endogenous Gata4 by short hairpin RNAs during reprogramming both accelerated and increased the efficiency of the process and augmented the mRNA levels of endogenous Nanog. Using comparative genomics, we identified a consensus binding site for Gata factors in an evolutionary conserved region located 9 kb upstream of the Nanog gene. Using chromatin immunoprecipitation, gel retardation, and luciferase assays, we found that Gata4 bound to this region and inhibited Nanog transcription in mouse embryonic stem cells. Overall, our results describe for first time the negative effect of Gata4 in the reprogramming of somatic cells and highlight the role of Gata factors in the transcriptional networks that control cell lineage choices in the early embryo.

A combination of transcriptomics and metabolomics uncovers enhanced bile acid biosynthesis in HepG2 cells expressing CCAAT/enhancer-binding protein ß (C/EBPß), hepatocyte nuclear factor 4α (HNF4α), and constitutive androstane receptor (CAR).

The development of hepatoma-based in vitro models to study hepatocyte physiology is an invaluable tool for both industry and academia. Here, we develop an in vitro model based on the HepG2 cell line that produces chenodeoxycholic acid, the main bile acid in humans, in amounts comparable to human hepatocytes. A combination of adenoviral transfections for CCAAT/enhancer-binding protein ß (C/EBPß), hepatocyte nuclear factor 4α (HNF4α), and constitutive androstane receptor (CAR) decreased intracellular glutamate, succinate, leucine, and valine levels in HepG2 cells, suggestive of a switch to catabolism to increase lipogenic acetyl CoA and increased anaplerosis to replenish the tricarboxylic acid cycle. Transcripts of key genes involved in bile acid synthesis were significantly induced by approximately 160-fold. Consistently, chenodeoxycholic acid production rate was increased by more than 20-fold. Comparison between mRNA and bile acid levels suggest that 12-alpha hydroxylation of 7-alpha-hydroxy-4-cholesten-3-one is the limiting step in cholic acid synthesis in HepG2 cells. These data reveal that introduction of three hepatocyte-related transcription factors enhance anabolic reactions in HepG2 cells and provide a suitable model to study bile acid biosynthesis under pathophysiological conditions.

Transcriptomic responses generated by hepatocarcinogens in a battery of liver-based in vitro models.

As the conventional approach to assess the potential of a chemical to cause cancer in humans still includes the 2-year rodent carcinogenicity bioassay, development of alternative methodologies is needed. In the present study, the transcriptomics responses following exposure to genotoxic (GTX) and non-genotoxic (NGTX) hepatocarcinogens and non-carcinogens (NC) in five liver-based in vitro models, namely conventional and epigenetically stabilized cultures of primary rat hepatocytes, the human hepatoma-derived cell lines HepaRG and HepG2 and human embryonic stem cell-derived hepatocyte-like cells, are examined. For full characterization of the systems, several bioinformatics approaches are employed including gene-based, ConsensusPathDB-based and classification analysis. They provide convincingly similar outcomes, namely that upon exposure to carcinogens, the HepaRG generates a gene classifier (a gene classifier is defined as a selected set of characteristic gene signatures capable of distinguishing GTX, NGTX carcinogens and NC) able to discriminate the GTX carcinogens from the NGTX carcinogens and NC. The other in vitro models also yield cancer-relevant characteristic gene groups for the GTX exposure, but some genes are also deregulated by the NGTX carcinogens and NC. Irrespective of the tested in vitro model, the most uniformly expressed pathways following GTX exposure are the p53 and those that are subsequently induced. The NGTX carcinogens triggered no characteristic cancer-relevant gene profiles in all liver-based in vitro systems. In conclusion, liver-based in vitro models coupled with transcriptomics techniques, especially in the case when the HepaRG cell line is used, represent valuable tools for obtaining insight into the mechanism of action and identification of GTX carcinogens.

A robust reprogramming strategy for generating hepatocyte-like cells usable in pharmaco-toxicological studies.

BACKGROUND: High-throughput pharmaco-toxicological testing frequently relies on the use of established liver-derived cell lines, such as HepG2 cells. However, these cells often display limited hepatic phenotype and features of neoplastic transformation that may bias the interpretation of the results. Alternate models based on primary cultures or differentiated pluripotent stem cells are costly to handle and difficult to implement in high-throughput screening platforms. Thus, cells without malignant traits, optimal differentiation pattern, producible in large and homogeneous amounts and with patient-specific phenotypes would be desirable. METHODS: We have designed and implemented a novel and robust approach to obtain hepatocytes from individuals by direct reprogramming, which is based on a combination of a single doxycycline-inducible polycistronic vector system expressing HNF4A, HNF1A and FOXA3, introduced in human fibroblasts previously transduced with human telomerase reverse transcriptase (hTERT). These cells can be maintained in fibroblast culture media, under standard cell culture conditions. RESULTS: Clonal hTERT-transduced human fibroblast cell lines can be expanded at least to 110 population doublings without signs of transformation or senescence. They can be easily differentiated at any cell passage number to hepatocyte-like cells with the simple addition of doxycycline to culture media. Acquisition of a hepatocyte phenotype is achieved in just 10 days and requires a simple and non-expensive cell culture media and standard 2D culture conditions. Hepatocytes reprogrammed from low and high passage hTERT-transduced fibroblasts display very similar transcriptomic profiles, biotransformation activities and show analogous pattern behavior in toxicometabolomic studies. Results indicate that this cell model outperforms HepG2 in toxicological screening. The procedure also allows generation of hepatocyte-like cells from patients with given pathological phenotypes. In fact, we succeeded in generating hepatocyte-like cells from a patient with alpha-1 antitrypsin deficiency, which recapitulated accumulation of intracellular alpha-1 antitrypsin polymers and deregulation of unfolded protein response and inflammatory networks. CONCLUSION: Our strategy allows the generation of an unlimited source of clonal, homogeneous, non-transformed induced hepatocyte-like cells, capable of performing typical hepatic functions and suitable for pharmaco-toxicological high-throughput testing. Moreover, as far as hepatocyte-like cells derived from fibroblasts isolated from patients suffering hepatic dysfunctions, retain the disease traits, as demonstrated for alpha-1-antitrypsin deficiency, this strategy can be applied to the study of other cases of anomalous hepatocyte functionality.

Modeling a Novel Variant of Glycogenosis IXa Using a Clonal Inducible Reprogramming System to Generate "Diseased" Hepatocytes for Accurate Diagnosis.

The diagnosis of inherited metabolic disorders is a long and tedious process. The matching of clinical data with a genomic variant in a specific metabolic pathway is an essential step, but the link between a genome and the clinical data is normally difficult, primarily for new missense variants or alterations in intron sequences. Notwithstanding, elucidation of the pathogenicity of a specific variant might be critical for an accurate diagnosis. In this study, we described a novel intronic variant c.2597 + 5G > T in the donor splice sequence of the PHKA2 gene. To investigate PHKA2 mRNA splicing, as well as the functional consequences on glycogen metabolism, we generated hepatocyte-like cells from a proband's fibroblasts by direct reprogramming. We demonstrated an aberrant splicing of PHKA2, resulting in the incorporation of a 27 bp upstream of intron 23 into exon 23, which leads to an immediate premature STOP codon. The truncated protein was unable to phosphorylate the PYGL protein, causing a 4-fold increase in the accumulation of glycogen in hepatocyte-like cells. Collectively, the generation of personalized hepatocyte-like cells enabled an unequivocal molecular diagnosis and qualified the sister's proband, a carrier of the same mutation, as a candidate for a preimplantation genetic diagnosis. Additionally, our direct reprogramming strategy allows for an unlimited source of "diseased" hepatocyte-like cells compatible with high-throughput platforms.

Derivation of healthy hepatocyte-like cells from a female patient with ornithine transcarbamylase deficiency through X-inactivation selection.

Autologous cell replacement therapy for inherited metabolic disorders requires the correction of the underlying genetic mutation in patient's cells. An unexplored alternative for females affected from X-linked diseases is the clonal selection of cells randomly silencing the X-chromosome containing the mutant allele, without in vivo or ex vivo genome editing. In this report, we have isolated dermal fibroblasts from a female patient affected of ornithine transcarbamylase deficiency and obtained clones based on inactivation status of either maternally or paternally inherited X chromosome, followed by differentiation to hepatocytes. Hepatocyte-like cells derived from these clones display indistinct features characteristic of hepatocytes, but express either the mutant or wild type OTC allele depending on X-inactivation pattern. When clonally derived hepatocyte-like cells were transplanted into FRG® KO mice, they were able to colonize the liver and recapitulate OTC-dependent phenotype conditioned by X-chromosome inactivation pattern. This approach opens new strategies for cell therapy of X-linked metabolic diseases and experimental in vitro models for drug development for such diseases.

Interaction between Hhex and SOX13 modulates Wnt/TCF activity.

Fine-tuning of the Wnt/TCF pathway is crucial for multiple embryological processes, including liver development. Here we describe how the interaction between Hhex (hematopoietically expressed homeobox) and SOX13 (SRY-related high mobility group box transcription factor 13), modulates Wnt/TCF pathway activity. Hhex is a homeodomain factor expressed in multiple endoderm-derived tissues, like the liver, where it is essential for proper development. The pleiotropic expression of Hhex during embryonic development and its dual role as a transcriptional repressor and activator suggest the presence of different tissue-specific partners capable of modulating its activity and function. While searching for developmentally regulated Hhex partners, we set up a yeast two-hybrid screening using an E9.5-10.5 mouse embryo library and the N-terminal domain of Hhex as bait. Among the putative protein interactors, we selected SOX13 for further characterization. We found that SOX13 interacts directly with full-length Hhex, and we delineated the interaction domains within the two proteins. SOX13 is known to repress Wnt/TCF signaling by interacting with TCF1. We show that Hhex is able to block the SOX13-dependent repression of Wnt/TCF activity by displacing SOX13 from the SOX13 x TCF1 complex. Moreover, Hhex de-repressed the Wnt/TCF pathway in the ventral foregut endoderm of cultured mouse embryos electroporated with a SOX13-expressing plasmid. We conclude that the interaction between Hhex and SOX13 may contribute to control Wnt/TCF signaling in the early embryo.

Direct conversion of human fibroblast to hepatocytes using a single inducible polycistronic vector.

BACKGROUND: Human fibroblasts can be reprogrammed into induced hepatocyte-like cells through the expression of a set of transcription factors. Although the generation of induced hepatocyte-like cells by HNF4A, HNF1A, and FOXA3 expression has proven to be a robust experimental strategy, using multiple lentivirus results in a highly variable heterogeneous population. METHODS: We designed and implemented a novel approach based on the delivery of reprogramming factors and green fluorescent protein in a single doxycycline-inducible lentiviral vector using 2A self-cleaving peptides. RESULTS: Fibroblasts infected with the lentiviral vector can be amplified in basic fibroblast culture media in the absence of doxycycline without induction of hepatic genes. Upon switching to hepatic maturation media containing doxycycline, cells stop proliferating, activate hepatic gene transcription, and perform metabolic functions characteristic of hepatocytes. CONCLUSION: Our strategy can generate an unlimited source of homogeneously induced hepatocyte-like cells from different genetic background donors, capable of performing typical hepatic functions suitable for drug research and other in vitro applications.

Glutamine/glutamate metabolism rewiring in reprogrammed human hepatocyte-like cells.

Human dermal fibroblasts can be reprogrammed into hepatocyte-like (HEP-L) cells by the expression of a set of transcription factors. Yet, the metabolic rewiring suffered by reprogrammed fibroblasts remains largely unknown. Here we report, using stable isotope-resolved metabolic analysis in combination with metabolomic-lipidomic approaches that HEP-L cells mirrors glutamine/glutamate metabolism in primary cultured human hepatocytes that is very different from parental human fibroblasts. HEP-L cells diverge glutamine from multiple metabolic pathways into deamidation and glutamate secretion, just like periportal hepatocytes do. Exceptionally, glutamine contribution to lipogenic acetyl-CoA through reductive carboxylation is increased in HEP-L cells, recapitulating that of primary cultured human hepatocytes. These changes can be explained by transcriptomic rearrangements of genes involved in glutamine/glutamate metabolism. Although metabolic changes in HEP-L cells are in line with reprogramming towards the hepatocyte lineage, our conclusions are limited by the fact that HEP-L cells generated do not display a complete mature phenotype. Nevertheless, our findings are the first to characterize metabolic adaptation in HEP-L cells that could ultimately be targeted to improve fibroblasts direct reprogramming to HEP-L cells.

The carcinoGENOMICS project: critical selection of model compounds for the development of omics-based in vitro carcinogenicity screening assays.

Recent changes in the European legislation of chemical-related substances have forced the scientific community to speed up the search for alternative methods that could partly or fully replace animal experimentation. The Sixth Framework Program project carcinoGENOMICS was specifically raised to develop omics-based in vitro screens for testing the carcinogenic potential of chemical compounds in a pan-European context. This paper provides an in-depth analysis of the complexity of choosing suitable reference compounds used for creating and fine-tuning the in vitro carcinogenicity assays. First, a number of solid criteria for the selection of the model compounds are defined. Secondly, the strategy followed, including resources consulted, is described and the selected compounds are briefly illustrated. Finally, limitations and problems encountered during the selection procedure are discussed. Since selecting an appropriate set of chemicals is a frequent impediment in the early stages of similar research projects, the information provided in this paper might be extremely valuable.

Silencing of hepatic fate-conversion factors induce tumorigenesis in reprogrammed hepatic progenitor-like cells.

BACKGROUND: Several studies have reported the direct conversion of mouse fibroblasts to hepatocyte-like cells with different degrees of maturation by expression of hepatic fate-conversion factors. METHODS: We have used a combination of lentiviral vectors expressing hepatic fate-conversion factors with Oct4, Sox2, Klf4, and Myc to convert mouse embryonic fibroblasts into hepatic cells. RESULTS: We have generated hepatic cells with progenitor-like features (iHepL cells). iHepL cells displayed basic hepatocyte functions but failed to perform functions characteristic of mature hepatocytes such as significant Cyp450 or urea cycle activities. iHepL cells expressed multiple hepatic-specific transcription factors and functional genes characteristic of immature hepatocytes and cholangiocytes, as well as high levels of Foxl1, Cd24a, and Lgr5, specific markers of hepatic progenitor cells. When transplanted into partial hepatectomized and hepatic irradiated mice, they differentiated into hepatocytes and cholangiocytes. However, iHepL cells formed malignant non-teratoma cell aggregations in one out of five engrafted livers and five out of five xenografts assays. All the cells in these tumors had silenced key hepatic fate-conversion factors, and lost hepatic features. CONCLUSIONS: This study highlights the dangers of using pluripotency factors in reprogramming strategies when fate-conversion factors are silenced in vivo, and urges us to perform extensive tumorigenic tests in reprogrammed cells.

Early ERK1/2 activation promotes DRP1-dependent mitochondrial fission necessary for cell reprogramming.

During the process of reprogramming to induced pluripotent stem (iPS) cells, somatic cells switch from oxidative to glycolytic metabolism, a transition associated with profound mitochondrial reorganization. Neither the importance of mitochondrial remodelling for cell reprogramming, nor the molecular mechanisms controlling this process are well understood. Here, we show that an early wave of mitochondrial fragmentation occurs upon expression of reprogramming factors. Reprogramming-induced mitochondrial fission is associated with a minor decrease in mitochondrial mass but not with mitophagy. The pro-fission factor Drp1 is phosphorylated early in reprogramming, and its knockdown and inhibition impairs both mitochondrial fragmentation and generation of iPS cell colonies. Drp1 phosphorylation depends on Erk activation in early reprogramming, which occurs, at least in part, due to downregulation of the MAP kinase phosphatase Dusp6. Taken together, our data indicate that mitochondrial fission controlled by an Erk-Drp1 axis constitutes an early and necessary step in the reprogramming process to pluripotency.

Dysfunctional mitochondrial fission impairs cell reprogramming.

We have recently shown that mitochondrial fission is induced early in reprogramming in a Drp1-dependent manner; however, the identity of the factors controlling Drp1 recruitment to mitochondria was unexplored. To investigate this, we used a panel of RNAi targeting factors involved in the regulation of mitochondrial dynamics and we observed that MiD51, Gdap1 and, to a lesser extent, Mff were found to play key roles in this process. Cells derived from Gdap1-null mice were used to further explore the role of this factor in cell reprogramming. Microarray data revealed a prominent down-regulation of cell cycle pathways in Gdap1-null cells early in reprogramming and cell cycle profiling uncovered a G2/M growth arrest in Gdap1-null cells undergoing reprogramming. High-Content analysis showed that this growth arrest was DNA damage-independent. We propose that lack of efficient mitochondrial fission impairs cell reprogramming by interfering with cell cycle progression in a DNA damage-independent manner.

Down-regulation of human CYP3A4 by the inflammatory signal interleukin-6: molecular mechanism and transcription factors involved.

The hepatic drug-metabolizing cytochrome P-450 (CYP) enzymes are down-regulated during inflammation. In vitro studies with hepatocytes have shown that the cytokines released during inflammatory responses are largely responsible for this CYP repression. However, the signaling pathways and the cytokine-activated factors involved remain to be properly identified. Our research has focused on the negative regulation of CYP3A4 (the major drug-metabolizing human CYP) by interleukin 6 (IL-6) (the principal regulator of the hepatic acute-phase response). CYP3A4 down-regulation by IL-6 requires activation of the glycoprotein receptor gp130; however, it does not proceed through the JAK/STAT pathway, as demonstrated by the overexpression of a dominant-negative STAT3 factor by means of an adenoviral vector. The involvement of IL-6-activated kinases such as extracellular signal-regulated kinase ERK1/2 or p38 is also unlikely, as evidenced by the use of specific chemical inhibitors. It is noteworthy that IL-6 caused a moderated induction in the mRNA of the transcription factor C/EBPbeta (CCAAT-enhancer binding protein beta) and a marked increase in the translation of C/EBPbeta-LIP, a 20-kDa C/EBPbeta isoform lacking a transactivation domain. Adenovirus-mediated expression of C/EBPbeta-LIP caused a dose-dependent repression of CYP3A4 mRNA, whereas overexpression C/EBPalpha and C/EBPb-LAP (35 kDa) caused a significant induction. Our results support the idea that IL-6 down-regulates CYP3A4 through translational induction of C/EBPbeta-LIP, which competes with and antagonizes constitutive C/EBP transactivators. From a clinical point of view, these findings could be relevant in the development of therapeutic cytokines with a less repressive effect on hepatic drug-metabolizing enzymes.

Transfection of Primary Hepatocytes with Liver-Enriched Transcription Factors Using Adenoviral Vectors.

Primary cultured hepatocytes are probably the best model to study endogenous metabolic pathways, toxicity, or drug metabolism. Many of these studies require expression of ectopic genes. It would be desirable to use a method of transfection that allows dose-response studies, high efficiency of transfection, and the possibility to express several genes at the same time. Adenoviral vectors fulfill these requirements, becoming a valuable tool for primary hepatocyte transfection. Moreover, they are easy to generate and do not require a high level of biocontainment. In the present chapter, we describe the generation, cloning, amplification, and purification of an adenoviral vector capable of infecting primary cultured hepatocytes. This recombinant adenovirus induces robust expression of the protein of interest in hepatocytes within a wide range of doses.

miR-122, a mammalian liver-specific microRNA, is processed from hcr mRNA and may downregulate the high affinity cationic amino acid transporter CAT-1.

These studies show that miR-122, a 22-nucleotide microRNA, is derived from a liver-specific noncoding polyadenylated RNA transcribed from the gene hcr. The exact sequence of miR-122 as well as the adjacent secondary structure within the hcr mRNA are conserved from mammalian species back to fish. Levels of miR-122 in the mouse liver increase to half maximal values around day 17 of embryogenesis, and reach near maximal levels of 50,000 copies per average cell before birth. Lewis et al. (2003) predicted the cationic amino acid transporter (CAT-1 or SLC7A1) as a miR-122 target. CAT-1 protein and its mRNA are expressed in all mammalian tissues but with lower levels in adult liver. Furthermore, during mouse liver development CAT-1 mRNA decreases in an almost inverse correlation with miR-122. Eight potential miR-122 target sites were predicted within the human CAT-1 mRNA, with six in the 3'-untranslated region. Using a reporter construct it was found that just three of the predicted sites, linked in a 400-nucleotide sequence from human CAT-1, acted with synergy and were sufficient to strongly inhibit protein synthesis and reduce mRNA levels. In summary, these studies followed the accumulation during development of miR-122 from its mRNA precursor, hcr, through to identification of what may be a specific mRNA target, CAT-1.

Maturation of induced pluripotent stem cell derived hepatocytes by 3D-culture.

Induced pluripotent stem cell derived hepatocytes (IPSC-Heps) have the potential to reduce the demand for a dwindling number of primary cells used in applications ranging from therapeutic cell infusions to in vitro toxicology studies. However, current differentiation protocols and culture methods produce cells with reduced functionality and fetal-like properties compared to adult hepatocytes. We report a culture method for the maturation of IPSC-Heps using 3-Dimensional (3D) collagen matrices compatible with high throughput screening. This culture method significantly increases functional maturation of IPSC-Heps towards an adult phenotype when compared to conventional 2D systems. Additionally, this approach spontaneously results in the presence of polarized structures necessary for drug metabolism and improves functional longevity to over 75 days. Overall, this research reveals a method to shift the phenotype of existing IPSC-Heps towards primary adult hepatocytes allowing such cells to be a more relevant replacement for the current primary standard.

Testing chemical carcinogenicity by using a transcriptomics HepaRG-based model?

The EU FP6 project carcinoGENOMICS explored the combination of toxicogenomics and in vitro cell culture models for identifying organotypical genotoxic- and non-genotoxic carcinogen-specific gene signatures. Here the performance of its gene classifier, derived from exposure of metabolically competent human HepaRG cells to prototypical non-carcinogens (10 compounds) and hepatocarcinogens (20 compounds), is reported. Analysis of the data at the gene and the pathway level by using independent biostatistical approaches showed a distinct separation of genotoxic from non-genotoxic hepatocarcinogens and non-carcinogens (up to 88 % correct prediction). The most characteristic pathway responding to genotoxic exposure was DNA damage. Interlaboratory reproducibility was assessed by blindly testing of three compounds, from the set of 30 compounds, by three independent laboratories. Subsequent classification of these compounds resulted in correct prediction of the genotoxicants. As expected, results on the non-genotoxic carcinogens and the non-carcinogens were less predictive. In conclusion, the combination of transcriptomics with the HepaRG in vitro cell model provides a potential weight of evidence approach for the evaluation of the genotoxic potential of chemical substances.

Hex homeobox gene controls the transition of the endoderm to a pseudostratified, cell emergent epithelium for liver bud development.

Little is known about the mechanism by which embryonic liver, lung, and pancreas progenitor cells emerge from the endodermal epithelium to initiate organogenesis. Understanding this process and its genetic control provides insight into ontogeny, developmental abnormalities, and tissue regeneration. We find that shortly after hepatic endoderm cells are specified, they undergo a transition from a columnar, gut morphology to a pseudostratified morphology, with concomitant "interkinetic nuclear migration" (INM) during cell division. INM is a hallmark of pseudostratified epithelia and the process used by neural progenitors to emerge from the neural epithelium. We find that the transition of the hepatic endoderm, but not the neural epithelium, to a pseudostratified epithelium is dependent upon the cell-autonomous activity of the homeobox gene Hex. In the absence of Hex, hepatic endoderm cells survive but maintain a columnar, simple epithelial phenotype and ectopically express Shh and other genes characteristic of the midgut epithelium. Thus, Hex promotes endoderm organogenesis by promoting the transition to a pseudostratified epithelium, which in turn allows hepatoblasts to emerge into the stromal environment and continue differentiating.