Zara Hannoun

Generation of functional human hepatic endoderm from human induced pluripotent stem cells

With the advent of induced pluripotent stem cell (iPSC) technology, it is now feasible to generate iPSCs with a defined genotype or disease state. When coupled with direct differentiation to a defined lineage, such as hepatic endoderm (HE), iPSCs would revolutionize the way we study human liver biology and generate efficient “off the shelf” models of human liver disease. Here, we show the “proof of concept” that iPSC lines representing both male and female sexes and two ethnic origins can be differentiated to HE at efficiencies of between 70%–90%, using a method mimicking physiological relevant condition. The iPSC‐derived HE exhibited hepatic morphology and expressed the hepatic markers albumin and E‐cadherin, as assessed by immunohistochemistry. They also expressed alpha‐fetoprotein, hepatocyte nuclear factor‐4a, and a metabolic marker, cytochrome P450 7A1 (Cyp7A1), demonstrating a definitive endodermal lineage differentiation. Furthermore, iPSC‐derived hepatocytes produced and secreted the plasma proteins, fibrinogen, fibronectin, transthyretin, and alpha‐fetoprotein, an essential feature for functional HE. Additionally iPSC‐derived HE supported both CYP1A2 and CYP3A4 metabolism, which is essential for drug and toxicology testing. Conclusion: This work is first to demonstrate the efficient generation of hepatic endodermal lineage from human iPSCs that exhibits key attributes of hepatocytes, and the potential application of iPSC‐derived HE in studying human liver biology. In particular, iPSCs from individuals representing highly polymorphic variants in metabolic genes and different ethnic groups will provide pharmaceutical development and toxicology studies a unique opportunity to revolutionize predictive drug toxicology assays and allow the creation of in vitro hepatic disease models. (HEPATOLOGY 2009.)

Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling

Human embryonic stem cells (hESCs) are a valuable source of pluripotential primary cells. To date, however, their homogeneous cellular differentiation to specific cell types in vitro has proven difficult. Wnt signaling has been shown to play important roles in coordinating development, and we demonstrate that Wnt3a is differentially expressed at critical stages of human liver development in vivo. The essential role of Wnt3a in hepatocyte differentiation from hESCs is paralleled by our in vitro model, demonstrating the importance of a physiologic approach to cellular differentiation. Our studies provide compelling evidence that Wnt3a signaling is important for coordinated hepatocellular function in vitro and in vivo. In addition, we demonstrate that Wnt3a facilitates clonal plating of hESCs exhibiting functional hepatic differentiation. These studies represent an important step toward the use of hESC-derived hepatocytes in high-throughput metabolic analysis of human liver function.

Lineage-specific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development

Methylation of cytosine is a DNA modification associated with gene repression. Recently, a novel cytosine modification, 5-hydroxymethylcytosine (5-hmC) has been discovered. Here we examine 5-hmC distribution during mammalian development and in cellular systems, and show that the developmental dynamics of 5-hmC are different from those of 5-methylcytosine (5-mC); in particular 5-hmC is enriched in embryonic contexts compared to adult tissues. A detectable 5-hmC signal appears in pre-implantation development starting at the zygote stage, where the paternal genome is subjected to a genome-wide hydroxylation of 5-mC, which precisely coincides with the loss of the 5-mC signal in the paternal pronucleus. Levels of 5-hmC are high in cells of the inner cell mass in blastocysts, and the modification colocalises with nestin-expressing cell populations in mouse post-implantation embryos. Compared to other adult mammalian organs, 5-hmC is strongly enriched in bone marrow and brain, wherein high 5-hmC content is a feature of both neuronal progenitors and post-mitotic neurons. We show that high levels of 5-hmC are not only present in mouse and human embryonic stem cells (ESCs) and lost during differentiation, as has been reported previously, but also reappear during the generation of induced pluripotent stem cells; thus 5-hmC enrichment correlates with a pluripotent cell state. Our findings suggest that apart from the cells of neuronal lineages, high levels of genomic 5-hmC are an epigenetic feature of embryonic cell populations and cellular pluri- and multi-lineage potency. To our knowledge, 5-hmC represents the first epigenetic modification of DNA discovered whose enrichment is so cell-type specific.

Post-translational modification by SUMO

Post-translational modifications (PTMs) are chemical alterations to a protein following translation, regulating stability and function. Reversible phosphorylation is an example of an important and well studied PTM involved in a number of cellular processes. SUMOylation is another PTM known to modify a large number of proteins and plays a role in various cellular processes including: cell cycle regulation, gene transcription, differentiation and cellular localisation. Therefore, understanding the role of SUMOylation in cell biology may allow the development of more efficient models, important in streamlining the drug discovery process. This review will focus on protein SUMOylation and its role in stem cell and somatic cell biology.

The Comparison between Conditioned Media and Serum-Free Media in Human Embryonic Stem Cell Culture and Differentiation

Human embryonic stem cells (hESCs) offer an inexhaustible supply of human somatic cell types through their ability to self-renew while retaining pluripotency. As such, hESC-derived cell types are important for applications ranging from in vitro modeling to therapeutic use. However, for their full potential to be realized, both the growth of the undifferentiated cells and their derivatives must be performed in defined culture conditions. Many research groups maintain hESCs using mouse embryonic fibroblasts (MEF) and MEF conditioned medium (CM). The use of murine systems to support hESCs has been imperative in developing hESC technology; however, they suffer from some major limitations including lack of definition, xenobiotic nature, batch-to-batch variation, and labor-intensive production. Therefore, hESC culture definition is essential if hESC lines, and their derivatives are to be quality assured and manufactured to GMP. We have initiated the process of standardizing hESC tissue culture and have employed two serum-free media: mTeSR (MT) and Stem Pro (SP). hESCs were maintained in a pluripotent state, for over 30 passages using MT and SP. Additionally, we present evidence that hESCs maintained in MT and SP generate equivalent levels of human hepatic endoderm as observed with CM. This data suggests that MT and SP are effective replacements for MEF-CM in hESC culture, contributing to the standardization of hESC in vitro models and ultimately their application.

The Inhibitory Role of Stromal Cell Mesenchyme on Human Embryonic Stem Cell Hepatocyte Differentiation is Overcome by Wnt3a Treatment

Pluripotent stem cells are derived from the inner cell mass of preimplantation embryos, and display the ability of the embryonic founder cells by forming all three germ lineages in vitro. It is well established that the cellular niche plays an important role in stem cell maintenance and differentiation. Stem cells generally have limited function without the specialized microenvironment of the niche that provides key cell-cell contact, soluble mediators, and extracellular matrices. We were interested in the role that Wnt signaling, in particular Wnt3a, played in human embryonic stem cell (hESC) differentiation to hepatic endoderm in vitro. hESC differentiation to hepatic endoderm was efficient in pure stem cell populations. However, in younger hESC lines, generating stromal cell mesenchyme, our model was very inefficient. The negative effect of stroma could be reversed by pretreating hESCs with Wnt3a prior to the onset of hepatocyte differentiation. Wnt3a pretreatment reinstated efficient hESC differentiation to hepatic endoderm. These studies represent an important step in understanding hepatocyte differentiation from hESCs and the role played by the cellular niche in vitro.

SUMOylation of HNF4α Regulates Protein Stability and Hepatocyte Function

Summary The coordination of signalling pathways within the cell is vital for normal human development and post-natal tissue homeostasis. Gene expression and function is therefore tightly controlled at a number of levels. We investigated the role that post-translational modifications play during human hepatocyte differentiation. In particular, we examined the role of the small ubiquitin-like modifier (SUMO) proteins in this process. We used a human embryonic stem cell (hESC)-based model of hepatocyte differentiation to follow changes in protein SUMOylation. Moreover, to confirm the results derived from our cell-based system, we performed in vitro conjugation assays to characterise SUMO modification of a key liver-enriched transcription factor, HNF4&agr;. Our analyses indicate that SUMOylation plays an important role during hepatocellular differentiation and this is mediated, in part, through regulation of the stability of HNF4&agr; in a ubiquitin-dependent manner. Our study provides a better understanding of SUMOylation during human hepatocyte differentiation and maturation. Moreover, we believe the results will stimulate interest in the differentiation and phenotypic regulation of other somatic cell types.

Hepatic Endoderm Differentiation from Human Embryonic Stem Cells

Primary human hepatocytes are a scarce resource with variable function which diminishes with time in culture. As a consequence their use in tissue modelling and therapy is restricted. Human embryonic stem cells (hESC) could provide a stable source of human tissue due to their properties of self-renewal and their ability to give rise to all three germ layers. hESCs have the potential to provide an unlimited supply of hepatic endoderm (HE) which could offer efficient tools for drug discovery, disease modelling and therapeutic applications. There has been a major focus on developing protocols to derive functional HE from hESCs. This review focuses on human liver biology and the translation of observations of in vivo systems into developing differentiation protocols to yield hepatic endoderm. It also details the potential role of oxygen tension as a new regulatory mechanism in HE differentiation and points out the importance of the mitochondrial function analysis in defining successful HE generation.

Deriving Metabolically Active Hepatic Endoderm from Pluripotent Stem Cells

The human liver is a vital organ within the body and plays a major role in normal homeostasis. The “work horse” of the liver, termed the “hepatocyte,” is estimated to make up approximately 70–80% of the liver’s mass. Therefore, the study of hepatocyte biology has an important role to play in medicine and the drug discovery process. At present the routine use of human primary hepatocytes is limited due to poor supply and their loss of function upon isolation. Therefore, additional and renewable sources of hepatocytes are being sought. Rodent hepatocytes have been utilised for many years, and although informative, they possess significant limitations and do not accurately extrapolate to human liver. To overcome the issue of cell viability, several groups have tried to generate immortalised hepatocytes; however, the derivative cells exhibit dramatic decreases in function and karyotypic instability over prolonged culture. It has therefore been necessary to find an alternative source of hepatocytes and efficient methods for deriving hepatic endoderm from stem cells in vitro. We have employed human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) to derive human hepatic endoderm (HE). hESCs and iPSCs represent scalable and highly efficient resources with which to generate human HE in vitro, and hESC-derived HE will be the focus of this chapter.

14-P024 Physiologic and defined directed differentiation of human embryonic stem cells and human endodermal cultures to hepatocytes

cephaly, as an adult it can cause brain atrophy and/or neurodegeneration. One common component of these disorders may be the disruption of neural stem cell (NSC) proliferation. Mouse NS-5 NSCs were treated for 48 h with basic fibroblast growth factor (10 ng/ml) or transforming growth factor (TGF) â1 (10 ng/ml) with or without ethanol (400 mg/dl). The effects of ethanol on cell cycle kinetics were examined with a cumulative bromodeoxyuridine labeling method. In the presence of either growth factor, ethanol increased cell cycle length through an elongated G1-phase. A comet assay for genomic integrity revealed that TGFâ1 or ethanol alone caused significant DNA damage (a 25% increase in the ‘‘tail”), and combined treatment was synergistic (‘‘tail” length was increased 125%). Total RNA was purified and analyzed on a Mouse 2.0 GeneChip (Affymetrix). The changes in cell cycle kinetics and DNA integrity were associated with alterations in DNA repairand cell cycle-transcript expression; of transcripts significantly affected by treatment played a role in cell cycle regulation or DNA damage response. Exemplary genes upor down-regulated 3-fold by TGFâ1 and/or ethanol were verified using reverse transcriptase polymerase chain reaction. An epigenetic study of CpG islands was performed to characterize patterns of DNA methylation. Many DNA synthesis and mitosis-related genes were hyper-methylated. Thus, ethanol restricts progression of NSCs through G1/S and G2/M checkpoints by differentially regulating the expression of genes involved in cell cycle control. Supported by NIAAA and the DVA.