FoxA factors: the chromatin key and doorstop essential for liver development and function.

Pioneer factors are transcriptional regulators with the capacity to bind inactive regions of chromatin and induce changes in accessibility that underpin cell fate decisions. The FOXA family of transcription factors is well understood to have pioneer capacity. Indeed, researchers have uncovered numerous examples of FOXA-dependent epigenomic modulation in developmental and disease processes. Despite the presence of FOXA being essential for correct epigenetic patterning, the need for continued FOXA presence postchromatin modulation has been debated. In a recent study in this issue of Genes & Development, Reizel and colleagues (pp. 1039-1050) show that the tissue-specific ablation of FOXA1/2/3 in the adult mouse liver results in the collapse of the epigenetic profile that maintains the hepatic gene expression profile. Thus, FOXA functions as a key, opening regions of chromatin during development, and as a doorstep, maintaining the established euchromatic structure in adult tissue.

GATA6 is a crucial factor for Myocd expression in the visceral smooth muscle cell differentiation program of the murine ureter.

Smooth muscle cells (SMCs) are a crucial component of the mesenchymal wall of the ureter, as they account for the efficient removal of the urine from the renal pelvis to the bladder by means of their contractile activity. Here, we show that the zinc-finger transcription factor gene Gata6 is expressed in mesenchymal precursors of ureteric SMCs under the control of BMP4 signaling. Mice with a conditional loss of Gata6 in these precursors exhibit a delayed onset and reduced level of SMC differentiation and peristaltic activity, as well as dilatation of the ureter and renal pelvis (hydroureternephrosis) at birth and at postnatal stages. Molecular profiling revealed a delayed and reduced expression of the myogenic driver gene Myocd, but the activation of signaling pathways and transcription factors previously implicated in activation of the visceral SMC program in the ureter was unchanged. Additional gain-of-function experiments suggest that GATA6 cooperates with FOXF1 in Myocd activation and SMC differentiation, possibly as pioneer and lineage-determining factors, respectively.

A PNPLA3-Deficient iPSC-Derived Hepatocyte Screen Identifies Pathways to Potentially Reduce Steatosis in Metabolic Dysfunction-Associated Fatty Liver Disease.

The incidence of nonalcoholic fatty liver disease (NAFLD), or metabolic dysfunction-associated fatty liver disease (MAFLD), is increasing in adults and children. Unfortunately, effective pharmacological treatments remain unavailable. Single nucleotide polymorphisms (SNPs) in the patatin-like phospholipase domain-containing protein (PNPLA3 I148M) have the most significant genetic association with the disease at all stages of its progression. A roadblock to identifying potential treatments for PNPLA3-induced NAFLD is the lack of a human cell platform that recapitulates the PNPLA3 I148M-mediated onset of lipid accumulation. Hepatocyte-like cells were generated from PNPLA3-/- and PNPLA3I148M/M-induced pluripotent stem cells (iPSCs). Lipid levels were measured by staining with BODIPY 493/503 and were found to increase in PNPLA3 variant iPSC-derived hepatocytes. A small-molecule screen identified multiple compounds that target Src/PI3K/Akt signaling and could eradicate lipid accumulation in these cells. We found that drugs currently in clinical trials for cancer treatment that target the same pathways also reduced lipid accumulation in PNPLA3 variant cells.

FGF2 mediates hepatic progenitor cell formation during human pluripotent stem cell differentiation by inducing the WNT antagonist NKD1.

Fibroblast growth factors (FGFs) are required to specify hepatic fate within the definitive endoderm through activation of the FGF receptors (FGFRs). While the signaling pathways involved in hepatic specification are well understood, the mechanisms through which FGFs induce hepatic character within the endoderm are ill defined. Here we report the identification of genes whose expression is directly regulated by FGFR activity during the transition from endoderm to hepatic progenitor cell. The FGFR immediate early genes that were identified include those encoding transcription factors, growth factors, and signaling molecules. One of these immediate early genes encodes naked cuticle homolog 1 (NKD1), which is a repressor of canonical WNT (wingless-type MMTV integration site) signaling. We show that loss of NKD1 suppresses the formation of hepatic progenitor cells from human induced pluripotent stem cells and that this phenotype can be rescued by using a pharmacological antagonist of canonical WNT signaling. We conclude that FGF specifies hepatic fate at least in large part by inducing expression of NKD1 to transiently suppress the canonical WNT pathway.

A small-molecule screen reveals that HSP90ß promotes the conversion of induced pluripotent stem cell-derived endoderm to a hepatic fate and regulates HNF4A turnover.

We have previously shown that the transcription factor HNF4A is required for the formation of hepatic progenitor cells from endoderm that has been derived from human induced pluripotent stem cells (iPSCs). We reasoned that we could uncover regulatory pathways with new roles in hepatocyte differentiation by identifying cellular processes that regulate HNF4A. We therefore performed a screen of 1120 small molecules with well-characterized mechanisms of action to detect those that affect the abundance of HNF4A in iPSC-derived hepatic progenitor cells. This approach uncovered several small molecules that depleted HNF4A. Of those, we chose to focus on an inhibitor of heat shock protein 90 beta (HSP90ß). We show that mutation of the gene encoding HSP90ß represses hepatocyte differentiation during the formation of hepatocytes from iPSCs. We reveal that HSP90ß, although dispensable for expression of HNF4A mRNA, directly interacts with HNF4A protein to regulate its half-life. Our results demonstrate that HSP90ß has an unappreciated role in controlling hepatic progenitor cell formation and highlight the efficiency of using small-molecule screens during the differentiation of iPSCs to reveal new molecular mechanisms that control hepatocyte formation.

The Use of Human Pluripotent Stem Cells for Modeling Liver Development and Disease.

The use of pluripotent stem cells (PSCs) has transformed the investigation of liver development and disease. Clinical observations and animal models have provided the foundations of our understanding in these fields. While animal models remain essential research tools, long experimental lead times and low throughput limit the scope of investigations. The ability of PSCs to produce large numbers of human hepatocyte-like cells, with a given or modified genetic background, allows investigators to use previously incompatible experimental techniques, such as high-throughput screens, to enhance our understanding of liver development and disease. In this review, we explore how PSCs have expedited our understanding of developmental mechanisms and have been used to identify new therapeutic options for numerous hepatic diseases. We discuss the future directions of the field, including how to further unlock the potential of the PSC model to make it amenable for use with a broader range of assays and a greater repertoire of diseases. Furthermore, we evaluate the current weaknesses of the PSC model and the directions open to researchers to address these limitations. Conclusion: The use of PSCs to model human liver disease and development has and will continue to have substantial impact, which is likely to further expand as protocols used to generate hepatic cells are improved.

Modeling Inborn Errors of Hepatic Metabolism Using Induced Pluripotent Stem Cells.

Inborn errors of hepatic metabolism are because of deficiencies commonly within a single enzyme as a consequence of heritable mutations in the genome. Individually such diseases are rare, but collectively they are common. Advances in genome-wide association studies and DNA sequencing have helped researchers identify the underlying genetic basis of such diseases. Unfortunately, cellular and animal models that accurately recapitulate these inborn errors of hepatic metabolism in the laboratory have been lacking. Recently, investigators have exploited molecular techniques to generate induced pluripotent stem cells from patients' somatic cells. Induced pluripotent stem cells can differentiate into a wide variety of cell types, including hepatocytes, thereby offering an innovative approach to unravel the mechanisms underlying inborn errors of hepatic metabolism. Moreover, such cell models could potentially provide a platform for the discovery of therapeutics. In this mini-review, we present a brief overview of the state-of-the-art in using pluripotent stem cells for such studies.

N-glycoprotein surfaceome of human induced pluripotent stem cell derived hepatic endoderm.

Using cell surface capture technology, the cell surface N-glycoproteome of human-induced pluripotent stem cell derived hepatic endoderm cells was assessed. Altogether, 395 cell surface N-glycoproteins were identified, represented by 1273 N-glycopeptides. This study identified N-glycoproteins that are not predicted to be localized to the cell surface and provides experimental data that assist in resolving ambiguous or incorrectly annotated transmembrane topology annotations. In a proof-of-concept analysis, combining these data with other cell surface proteome datasets is useful for identifying potentially cell type and lineage restricted markers and drug targets to advance the use of stem cell technologies for mechanistic developmental studies, disease modeling, drug discovery, and regenerative medicine.

Epicardial GATA factors regulate early coronary vascular plexus formation.

During early development, GATA factors have been shown to be important for key events of coronary vasculogenesis, including formation of the epicardium. Myocardial GATA factors are required for coronary vascular (CV) formation; however, the role of epicardial localized GATAs in this process has not been addressed. The current study was conducted to investigate the molecular mechanisms by which the epicardium controls coronary vasculogenesis, focusing on the role of epicardial GATAs in establishing the endothelial plexus during early coronary vasculogenesis. To address the role of epicardial GATAs, we ablated GATA4 and GATA6 transcription factors specifically from the mouse epicardium and found that the number of endothelial cells in the sub-epicardium was drastically reduced, and concomitant coronary vascular plexus formation was significantly compromised. Here we present evidence for a novel role for epicardial GATA factors in controlling plexus formation by recruiting endothelial cells to the sub-epicardium.

Identification of small molecules for human hepatocyte expansion and iPS differentiation.

Cell-based therapies hold the potential to alleviate the growing burden of liver diseases. Such therapies require human hepatocytes, which, within the stromal context of the liver, are capable of many rounds of replication. However, this ability is lost ex vivo, and human hepatocyte sourcing has limited many fields of research for decades. Here we developed a high-throughput screening platform for primary human hepatocytes to identify small molecules in two different classes that can be used to generate renewable sources of functional human hepatocytes. The first class induced functional proliferation of primary human hepatocytes in vitro. The second class enhanced hepatocyte functions and promoted the differentiation of induced pluripotent stem cell-derived hepatocytes toward a more mature phenotype than what was previously obtainable. The identification of these small molecules can help address a major challenge affecting many facets of liver research and may lead to the development of new therapeutics for liver diseases.

Modeling hepatitis C virus infection using human induced pluripotent stem cells.

Human pathogens impact patient health through a complex interplay with the host, but models to study the role of host genetics in this process are limited. Human induced pluripotent stem cells (iPSCs) offer the ability to produce host-specific differentiated cells and thus have the potential to transform the study of infectious disease; however, no iPSC models of infectious disease have been described. Here we report that hepatocyte-like cells derived from iPSCs support the entire life cycle of hepatitis C virus, including inflammatory responses to infection, enabling studies of how host genetics impact viral pathogenesis.

HNF4A is essential for specification of hepatic progenitors from human pluripotent stem cells.

The availability of pluripotent stem cells offers the possibility of using such cells to model hepatic disease and development. With this in mind, we previously established a protocol that facilitates the differentiation of both human embryonic stem cells and induced pluripotent stem cells into cells that share many characteristics with hepatocytes. The use of highly defined culture conditions and the avoidance of feeder cells or embryoid bodies allowed synchronous and reproducible differentiation to occur. The differentiation towards a hepatocyte-like fate appeared to recapitulate many of the developmental stages normally associated with the formation of hepatocytes in vivo. In the current study, we addressed the feasibility of using human pluripotent stem cells to probe the molecular mechanisms underlying human hepatocyte differentiation. We demonstrate (1) that human embryonic stem cells express a number of mRNAs that characterize each stage in the differentiation process, (2) that gene expression can be efficiently depleted throughout the differentiation time course using shRNAs expressed from lentiviruses and (3) that the nuclear hormone receptor HNF4A is essential for specification of human hepatic progenitor cells by establishing the expression of the network of transcription factors that controls the onset of hepatocyte cell fate.

A cell surfaceome map for immunophenotyping and sorting pluripotent stem cells.

Induction of a pluripotent state in somatic cells through nuclear reprogramming has ushered in a new era of regenerative medicine. Heterogeneity and varied differentiation potentials among induced pluripotent stem cell (iPSC) lines are, however, complicating factors that limit their usefulness for disease modeling, drug discovery, and patient therapies. Thus, there is an urgent need to develop nonmutagenic rapid throughput methods capable of distinguishing among putative iPSC lines of variable quality. To address this issue, we have applied a highly specific chemoproteomic targeting strategy for de novo discovery of cell surface N-glycoproteins to increase the knowledge-base of surface exposed proteins and accessible epitopes of pluripotent stem cells. We report the identification of 500 cell surface proteins on four embryonic stem cell and iPSCs lines and demonstrate the biological significance of this resource on mouse fibroblasts containing an oct4-GFP expression cassette that is active in reprogrammed cells. These results together with immunophenotyping, cell sorting, and functional analyses demonstrate that these newly identified surface marker panels are useful for isolating iPSCs from heterogeneous reprogrammed cultures and for isolating functionally distinct stem cell subpopulations.

Endoplasmic reticulum-tethered transcription factor cAMP responsive element-binding protein, hepatocyte specific, regulates hepatic lipogenesis, fatty acid oxidation, and lipolysis upon metabolic stress in mice.

UNLABELLED: cAMP responsive element-binding protein, hepatocyte specific (CREBH), is a liver-specific transcription factor localized in the endoplasmic reticulum (ER) membrane. Our previous work demonstrated that CREBH is activated by ER stress or inflammatory stimuli to induce an acute-phase hepatic inflammation. Here, we demonstrate that CREBH is a key metabolic regulator of hepatic lipogenesis, fatty acid (FA) oxidation, and lipolysis under metabolic stress. Saturated FA, insulin signals, or an atherogenic high-fat diet can induce CREBH activation in the liver. Under the normal chow diet, CrebH knockout mice display a modest decrease in hepatic lipid contents, but an increase in plasma triglycerides (TGs). After having been fed an atherogenic high-fat (AHF) diet, massive accumulation of hepatic lipid metabolites and significant increase in plasma TG levels were observed in the CrebH knockout mice. Along with the hypertriglyceridemia phenotype, the CrebH null mice displayed significantly reduced body-weight gain, diminished abdominal fat, and increased nonalcoholic steatohepatitis activities under the AHF diet. Gene-expression analysis and chromatin-immunoprecipitation assay indicated that CREBH is required to activate the expression of the genes encoding functions involved in de novo lipogenesis, TG and cholesterol biosynthesis, FA elongation and oxidation, lipolysis, and lipid transport. Supporting the role of CREBH in lipogenesis and lipolysis, forced expression of an activated form of CREBH protein in the liver significantly increases accumulation of hepatic lipids, but reduces plasma TG levels in mice. CONCLUSION: All together, our study shows that CREBH plays a key role in maintaining lipid homeostasis by regulating the expression of the genes involved in hepatic lipogenesis, FA oxidation, and lipolysis under metabolic stress. The identification of CREBH as a stress-inducible metabolic regulator has important implications in the understanding and treatment of metabolic disease.

JD induced pluripotent stem cell-derived hepatocytes faithfully recapitulate the pathophysiology of familial hypercholesterolemia.

UNLABELLED: Elevated levels of low-density lipoprotein cholesterol (LDL-C) in plasma are a major contributor to cardiovascular disease, which is the leading cause of death worldwide. Genome-wide association studies (GWAS) have identified 95 loci that associate with control of lipid/cholesterol metabolism. Although GWAS results are highly provocative, direct analyses of the contribution of specific allelic variations in regulating LDL-C has been challenging due to the difficulty in accessing appropriate cells from affected patients. The primary cell type responsible for controlling cholesterol and lipid flux is the hepatocyte. Recently, we have shown that cells with hepatocyte characteristics can be generated from human induced pluripotent stem cells (iPSCs). This finding raises the possibility of using patient-specific iPSC-derived hepatocytes to study the functional contribution of GWAS loci in regulating lipid metabolism. To test the validity of this approach, we produced iPSCs from JD a patient with mutations in the low-density lipoprotein receptor (LDLR) gene that result in familial hypercholesterolemia (FH). We demonstrate that (1) hepatocytes can be efficiently generated from FH iPSCs; (2) in contrast to control cells, FH iPSC-derived hepatocytes are deficient in LDL-C uptake; (3) control but not FH iPSC-derived hepatocytes increase LDL uptake in response to lovastatin; and (4) FH iPSC-derived hepatocytes display a marked elevation in secretion of lipidated apolipoprotein B-100. CONCLUSION: Cumulatively, these findings demonstrate that FH iPSC-derived hepatocytes recapitulate the complex pathophysiology of FH in culture. These results also establish that patient-specific iPSC-derived hepatocytes could be used to definitively determine the functional contribution of allelic variation in regulating lipid and cholesterol metabolism and could potentially provide a platform for the identification of novel treatments of cardiovascular disease. (HEPATOLOGY 2012).

Hepatocyte nuclear factor 4alpha controls the development of a hepatic epithelium and liver morphogenesis.

Although advances have been made in understanding cell differentiation, only rudimentary knowledge exists concerning how differentiated cells form tissues and organs. We studied liver organogenesis because the cell and tissue architecture of this organ is well defined. Approximately 60% of the adult liver consists of hepatocytes that are arranged as single-cell anastomosing plates extending from the portal region of the liver lobule toward the central vein. The basal surface of the hepatocytes is separated from adjacent sinusoidal endothelial cells by the space of Disse, where the exchange of substances between serum and hepatocytes takes place. The hepatocyte's apical surface forms bile canaliculi that transport bile to the hepatic ducts. Proper liver architecture is crucial for hepatic function and is commonly disrupted in disease states, including cirrhosis and hepatitis. Here we report that hepatocyte nuclear factor 4alpha (Hnf4alpha) is essential for morphological and functional differentiation of hepatocytes, accumulation of hepatic glycogen stores and generation of a hepatic epithelium. We show that Hnf4alpha is a dominant regulator of the epithelial phenotype because its ectopic expression in fibroblasts induces a mesenchymal-to-epithelial transition. Most importantly, the morphogenetic parameters controlled by Hnf4alpha in hepatocytes are essential for normal liver architecture, including the organization of the sinusoidal endothelium.

CDK5-PRMT1-WDR24 signaling cascade promotes mTORC1 signaling and tumor growth.

The mammalian target of rapamycin complex1 (mTORC1) is a central regulator of metabolism and cell growth by sensing diverse environmental signals, including amino acids. The GATOR2 complex is a key component linking amino acid signals to mTORC1. Here, we identify protein arginine methyltransferase 1 (PRMT1) as a critical regulator of GATOR2. In response to amino acids, cyclin-dependent kinase 5 (CDK5) phosphorylates PRMT1 at S307 to promote PRMT1 translocation from nucleus to cytoplasm and lysosome, which in turn methylates WDR24, an essential component of GATOR2, to activate the mTORC1 pathway. Disruption of the CDK5-PRMT1-WDR24 axis suppresses hepatocellular carcinoma (HCC) cell proliferation and xenograft tumor growth. High PRMT1 protein expression is associated with elevated mTORC1 signaling in patients with HCC. Thus, our study dissects a phosphorylation- and arginine methylation-dependent regulatory mechanism of mTORC1 activation and tumor growth and provides a molecular basis to target this pathway for cancer therapy.

A human iPSC-derived hepatocyte screen identifies compounds that inhibit production of Apolipoprotein B.

Familial hypercholesterolemia (FH) patients suffer from excessively high levels of Low Density Lipoprotein Cholesterol (LDL-C), which can cause severe cardiovascular disease. Statins, bile acid sequestrants, PCSK9 inhibitors, and cholesterol absorption inhibitors are all inefficient at treating FH patients with homozygous LDLR gene mutations (hoFH). Drugs approved for hoFH treatment control lipoprotein production by regulating steady-state Apolipoprotein B (apoB) levels. Unfortunately, these drugs have side effects including accumulation of liver triglycerides, hepatic steatosis, and elevated liver enzyme levels. To identify safer compounds, we used an iPSC-derived hepatocyte platform to screen a structurally representative set of 10,000 small molecules from a proprietary library of 130,000 compounds. The screen revealed molecules that could reduce the secretion of apoB from cultured hepatocytes and from humanized livers in mice. These small molecules are highly effective, do not cause abnormal lipid accumulation, and share a chemical structure that is distinct from any known cholesterol lowering drug.

GATA factors regulate proliferation, differentiation, and gene expression in small intestine of mature mice.

BACKGROUND & AIMS: GATA transcription factors regulate proliferation, differentiation, and gene expression in multiple organs. GATA4 is expressed in the proximal 85% of the small intestine and regulates the jejunal-ileal gradient in absorptive enterocyte gene expression. GATA6 is co-expressed with GATA4 but also is expressed in the ileum; its function in the mature small intestine is unknown. METHODS: We investigated the function of GATA6 in small intestine using adult mice with conditional, inducible deletion of Gata6, or Gata6 and Gata4, specifically in the intestine. RESULTS: In ileum, deletion of Gata6 caused a decrease in crypt cell proliferation and numbers of enteroendocrine and Paneth cells, an increase in numbers of goblet-like cells in crypts, and altered expression of genes specific to absorptive enterocytes. In contrast to ileum, deletion of Gata6 caused an increase in numbers of Paneth cells in jejunum and ileum. Deletion of Gata6 and Gata4 resulted in a jejunal and duodenal phenotype that was nearly identical to that in the ileum after deletion of Gata6 alone, revealing common functions for GATA6 and GATA4. CONCLUSIONS: GATA transcription factors are required for crypt cell proliferation, secretory cell differentiation, and absorptive enterocyte gene expression in the small intestinal epithelium.