Stephen A. Duncan

Highly efficient generation of human hepatocyte–like cells from induced pluripotent stem cells

There exists a worldwide shortage of donor livers available for orthotropic liver transplantation and hepatocyte transplantation therapies. In addition to their therapeutic potential, primary human hepatocytes facilitate the study of molecular and genetic aspects of human hepatic disease and development and provide a platform for drug toxicity screens and identification of novel pharmaceuticals with potential to treat a wide array of metabolic diseases. The demand for human hepatocytes, therefore, heavily outweighs their availability. As an alternative to using donor livers as a source of primary hepatocytes, we explored the possibility of generating patient‐specific human hepatocytes from induced pluripotent stem (iPS) cells. Conclusion: We demonstrate that mouse iPS cells retain full potential for fetal liver development and describe a procedure that facilitates the efficient generation of highly differentiated human hepatocyte‐like cells from iPS cells that display key liver functions and can integrate into the hepatic parenchyma in vivo. (HEPATOLOGY 2010.)

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 hepatocytes 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 4α (Hnf4α) is essential for morphological and functional differentiation of hepatocytes, accumulation of hepatic glycogen stores and generation of a hepatic epithelium. We show that Hnf4α 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 Hnf4α in hepatocytes are essential for normal liver architecture, including the organization of the sinusoidal endothelium.

Organogenesis and development of the liver.

Embryonic development of the liver has been studied intensely, yielding insights that impact diverse areas of developmental and cell biology. Understanding the fundamental mechanisms that control hepatogenesis has also laid the basis for the rational differentiation of stem cells into cells that display many hepatic functions. Here, we review the basic molecular mechanisms that control the formation of the liver as an organ.

The orphan nuclear receptor HNF4α determines PXR- and CAR-mediated xenobiotic induction of CYP3A4

The drug metabolizing enzyme cytochrome P450 3A4 (CYP3A4) is thought to be involved in the metabolism of nearly 50% of all the drugs currently prescribed. Alteration in the activity or expression of this enzyme seems to be a key predictor of drug responsiveness and toxicity. Currently available studies indicate that the ligand-activated nuclear receptors pregnane X receptor (PXR; NR1I2) and constitutive androstane receptor (CAR; NR1I3) regulate CYP3A4 expression. However, in cell-based reporter assays, CYP3A4 promoter activity was most pronounced in liver-derived cells and minimal or modest in non-hepatic cells, indicating that a liver-specific factor is required for physiological transcriptional response. Here we show that the orphan nuclear receptor hepatocyte nuclear factor-4α (HNF4α; HNF4A) is critically involved in the PXR- and CAR-mediated transcriptional activation of CYP3A4. We identified a specific cis-acting element in the CYP3A4 gene enhancer that confers HNF4α binding and thereby permits PXR- and CAR-mediated gene activation. Fetal mice with conditional deletion of Hnf4α had reduced or absent expression of CYP3A. Furthermore, adult mice with conditional hepatic deletion of Hnf4α had reduced basal and inducible expression of CYP3A. These data identify HNF4α as an important regulator of coordinate nuclear-receptor–mediated response to xenobiotics.

Cardiac-Specific Deletion of Gata4 Reveals Its Requirement for Hypertrophy, Compensation, and Myocyte Viability

The transcription factor GATA4 is a critical regulator of cardiac gene expression where it controls embryonic development, cardiomyocyte differentiation, and stress responsiveness of the adult heart. Traditional deletion of Gata4 caused embryonic lethality associated with endoderm defects and cardiac malformations, precluding an analysis of the role of GATA4 in the adult myocardium. To address the function of GATA4 in the adult heart, Gata4-loxP–targeted mice (Gata4fl/fl) were crossed with mice containing a β-myosin heavy chain (β-MHC) or α-MHC promoter-driven Cre transgene, which produced viable mice that survived into adulthood despite a 95% and 70% loss of GATA4 protein, respectively. However, cardiac-specific deletion of Gata4 resulted in a progressive and dosage-dependent deterioration in cardiac function and dilation in adulthood. Moreover, pressure overload stimulation induced rapid decompensation and heart failure in cardiac-specific Gata4-deleted mice. More provocatively, Gata4-deleted mice were compromised in their ability to hypertrophy following pressure overload or exercise stimulation. Mechanistically, cardiac-specific deletion of Gata4 increased cardiomyocyte TUNEL at baseline in embryos and adults as they aged, as well as dramatically increased TUNEL following pressure overload stimulation. Examination of gene expression profiles in the heart revealed a number of profound alterations in known GATA4-regulated structural genes as well as genes with apoptotic implications. Thus, GATA4 is a necessary regulator of cardiac gene expression, hypertrophy, stress-compensation, and myocyte viability.

ER Stress Controls Iron Metabolism Through Induction of Hepcidin

Ironing Out Stress The peptide hormone, hepcidin, is secreted from the liver in response to extracellular factors, including inflammation, and regulates iron homeostasis by controlling transmembrane iron transport. Vecchi et al. (p. 877) showed that intracellular stress signals in the endoplasmic reticulum also control hepcidin expression and can thus modulate local or systemic iron traffic. This mechanism occurs through the transcription factor CREBH, which is a known mediator of the inflammatory response. Collectively, the results suggest a direct link between the intracellular stress response, innate immunity, and iron metabolism. Stress signals in the endoplasmic reticulum activate a transcription factor that induces the expression of an iron-regulatory hormone. Hepcidin is a peptide hormone that is secreted by the liver and controls body iron homeostasis. Hepcidin overproduction causes anemia of inflammation, whereas its deficiency leads to hemochromatosis. Inflammation and iron are known extracellular stimuli for hepcidin expression. We found that endoplasmic reticulum (ER) stress also induces hepcidin expression and causes hypoferremia and spleen iron sequestration in mice. CREBH (cyclic AMP response element–binding protein H), an ER stress–activated transcription factor, binds to and transactivates the hepcidin promoter. Hepcidin induction in response to exogenously administered toxins or accumulation of unfolded protein in the ER is defective in CREBH knockout mice, indicating a role for CREBH in ER stress–regulated hepcidin expression. The regulation of hepcidin by ER stress links the intracellular response involved in protein quality control to innate immunity and iron homeostasis.

Embryonic development of the liver

The combined use of genetic manipulation in mice and advanced culturing techniques, along with the availability of model genetic systems, underlies the recent expansion in our understanding of the fundamental processes that govern the development of the liver. Although gaps in our knowledge still exist, many of the molecules and pathways that act during the earliest stages of hepatic development have been identified. This knowledge has been applied to control the differentiation of embryonic stem and hepatic progenitor cells into hepatocytes and cholangiocytes, raising the prospect of generating quantities of hepatic cells in culture that are suitable for therapeutic uses.

GATA6 Is Essential for Embryonic Development of the Liver but Dispensable for Early Heart Formation

ABSTRACT Several lines of evidence suggest that GATA6 has an integral role in controlling development of the mammalian liver. Unfortunately, this proposal has been impossible to address directly because mouse embryos lacking GATA6 die during gastrulation. Here we show that the early embryonic deficiency associated with GATA6-knockout mice can be overcome by providing GATA6-null embryos with a wild-type extraembryonic endoderm with the use of tetraploid embryo complementation. Analysis of rescued Gata6 − / − embryos revealed that, although hepatic specification occurs normally, the specified cells fail to differentiate and the liver bud does not expand. Although GATA6 is expressed in multiple tissues that impact development of the liver, including the heart, septum transversum mesenchyme, and vasculature, all are relatively unaffected by loss of GATA6, which is consistent with a cell-autonomous requirement for GATA6 during hepatogenesis. We also demonstrate that a closely related GATA factor, GATA4, is expressed transiently in the prehepatic endoderm during hepatic specification and then lost during expansion of the hepatic primordium. Our data support the proposal that GATA4 and GATA6 are functionally redundant during hepatic specification but that GATA6 alone is available for liver bud growth and commitment of the endoderm to a hepatic cell fate.

The MODY1 gene HNF-4α regulates selected genes involved in insulin secretion

Mutations in the gene encoding hepatocyte nuclear factor-4alpha (HNF-4alpha) result in maturity-onset diabetes of the young (MODY). To determine the contribution of HNF-4alpha to the maintenance of glucose homeostasis by the beta cell in vivo, we derived a conditional knockout of HNF-4alpha using the Cre-loxP system. Surprisingly, deletion of HNF-4alpha in beta cells resulted in hyperinsulinemia in fasted and fed mice but paradoxically also in impaired glucose tolerance. Islet perifusion and calcium-imaging studies showed abnormal responses of the mutant beta cells to stimulation by glucose and sulfonylureas. These phenotypes can be explained in part by a 60% reduction in expression of the potassium channel subunit Kir6.2. We demonstrate using cotransfection assays that the Kir6.2 gene is a transcriptional target of HNF-4alpha. Our data provide genetic evidence that HNF-4alpha is required in the pancreatic beta cell for regulation of the pathway of insulin secretion dependent on the ATP-dependent potassium channel.

Cardiomyocyte GATA4 functions as a stress-responsive regulator of angiogenesis in the murine heart

The transcription factor GATA4 is a critical regulator of cardiac gene expression, modulating cardiomyocyte differentiation and adaptive responses of the adult heart. We report what we believe to be a novel function for GATA4 in murine cardiomyocytes as a nodal regulator of cardiac angiogenesis. Conditional overexpression of GATA4 within adult cardiomyocytes increased myocardial capillary and small conducting vessel densities and increased coronary flow reserve and perfusion-dependent cardiac contractility. Coculture of HUVECs with either GATA4-expressing cardiomyocytes or with myocytes expressing a dominant-negative form of GATA4 enhanced or reduced HUVEC tube formation, respectively. Expression of GATA4 in skeletal muscle by adenoviral gene transfer enhanced capillary densities and hindlimb perfusion following femoral artery ablation. Deletion of Gata4 specifically from cardiomyocytes reduced myocardial capillary density and prevented pressure overload-augmented angiogenesis in vivo. GATA4 induced the angiogenic factor VEGF-A, directly binding the Vegf-A promoter and enhancing transcription. GATA4-overexpressing mice showed increased levels of cardiac VEGF-A, while Gata4-deleted mice demonstrated decreased VEGF-A levels. The induction of HUVEC tube formation in GATA4-overexpressing cocultured myocytes was blocked with a VEGF receptor antagonist. Pressure overload-induced dysfunction in Gata4-deleted hearts was partially rescued by adenoviral gene delivery of VEGF and angiopoietin-1. To our knowledge, these results demonstrate [corrected] a previously unrecognized function for GATA4 as a regulator of cardiac angiogenesis through a nonhypoxic, load, and/or disease-responsive mechanism.