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In JoVE (2)

Other Publications (23)

Articles by David C. Hay in JoVE

 JoVE Biology

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

1Medical Research Council Centre for Regenerative Medicine, University of Edinburgh


JoVE 2969

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

 JoVE Chemistry

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

1MRC Centre for Regenerative Medicine, University of Edinburgh, 2School of Chemistry, University of Edinburgh, 3MRC Centre for Inflammation Research, University of Edinburgh


JoVE 51723

This article will focus on developing polymer coated surfaces for long-term, stable culture of stem cell derived human hepatocytes.

Other articles by David C. Hay on PubMed

Activation of NF-kappaB Nuclear Transcription Factor by Flow in Human Endothelial Cells

The tractive force generated by blood flow, called fluid shear stress, is an important regulator of endothelial cell gene expression. Several transcription factors are activated by shear stress, including members of the NF-kappaB/Rel family. The nature of the upstream-signaling components involved in the activation of NF-kappaB by flow has been studied in human endothelial cells. Flow rapidly increased endogenous IKK1/2 activity and transiently degraded IkappaBalpha and IkappaBbeta1, but not p105/p50. Nuclear translocation of the p65 subunit was induced by flow in wild-type (w/t) cells and in cells overexpressing w/t NIK, IKK1 or IKK2, but not in cells transiently transfected with kinase-inactive mutants of these enzymes. Nuclear translocation of p65 in response to flow was not affected by overexpressing a dominant-negative mutant of a MAPKKK related to NIK, called TPL2 kinase, nor by pretreating cells with the selective PKC inhibitor bisindoylmaleimide-1. Gel shift assays showed that the binding of p50/p65 heterodimer to radiolabeled oligonucleotide containing a shear-stress response element was increased by flow. The activity of a 3kappaB conA-luciferase reporter was also increased, confirming that NF-kappaB activated by flow was transcriptionally active. We conclude that shear stress induces gene transactivation by NF-kappaB (p50/p65) via the NIK-IKK1/2 pathway and proteosome-dependent degradation of IkappaB and that induction by flow does not involve TPL-2 kinase or PKC.

Oct-4 Knockdown Induces Similar Patterns of Endoderm and Trophoblast Differentiation Markers in Human and Mouse Embryonic Stem Cells

The transcription factor Oct-4 is a marker of pluripotency in mouse and human embryonic stem (ES) cells. Previous studies using a tetracycline-regulated Oct-4 transgene in the ZHBTc4 cell line demonstrated that downregulation of Oct-4 expression induced dedifferentiation into trophoblast, a lineage mouse ES cells do not normally generate. We found that transfection of Oct-4-specific short interfering RNA significantly reduced expression and functional activity of Oct-4 in mouse and human ES cells, enabling its role to be compared in both cell types. In mouse ES cells, Oct-4 knockdown produced a pattern of morphological differentiation and increase in expression of the trophoblast-associated transcription factor Cdx2, similar to that triggered by suppressing the Oct-4 transgene in the ZHBTc4 cell line. In addition, downregulation of Oct-4 was accompanied by increased expression of the endoderm-associated genes Gata6 and alpha-fetoprotein, and a gene trap associated with primitive liver/yolk sac differentiation. In human ES cells, Oct-4 knockdown also induced morphological differentiation coincident with the upregulation of Gata6. The induction of Cdx2 and other trophoblast-associated genes, however, was dependent on the culture conditions. These results establish the general requirement for Oct-4 in maintaining pluripotency in ES cells. Moreover, the upregulation of endoderm-associated markers in both mouse and human ES cells points to overlap between development of trophoblast and endoderm differentiation.

Direct Differentiation of Human Embryonic Stem Cells to Hepatocyte-like Cells Exhibiting Functional Activities

The utilization of human hepatocytes for biomedical research, drug discovery, and treatment of liver diseases is hindered by the limited availability of donated livers and the variability of their derived hepatocytes. Human embryonic stem cells (hESCs) are pluripotent and provide a unique, unlimited resource for human hepatocytes. However, differentiation of hESCs to hepatocytes remains a challenge. We have developed a multistage procedure by which hESCs can be directly differentiated to hepatocyte-like cells without embryoid body formation and the requirement of sodium butyrate. The hESC-derived hepatocyte-like cells (HLCs) exhibited characteristic hepatocyte morphology, expressed hepatocyte markers, including alpha-fetoprotein, albumin, and hepatocyte nuclear factor 4alpha, and possessed hepatocyte-specific activities, such as p450 metabolism, albumin production, glycogen storage, and uptake and excretion of indocyanine green. Hepatocyte growth factor was found to play a positive role in promoting hepatocyte differentiation. Our differentiation system has shown that hESCs can be differentiated to hepatocyte-like cells capable of executing a range of hepatocyte functions. Therefore, it presents a proof-of-principle of potential applications of using the hESC-derived hepatocytes. Additionally, the hESC-derived HLCs provide a unique model to study the mechanisms involved in human hepatocyte differentiation and liver function.

Efficient Differentiation of Hepatocytes from Human Embryonic Stem Cells Exhibiting Markers Recapitulating Liver Development in Vivo

The potential to differentiate human embryonic stem cells (hESCs) in vitro to provide an unlimited source of human hepatocytes for use in biomedical research, drug discovery, and the treatment of liver diseases holds great promise. Here we describe a three-stage process for the efficient and reproducible differentiation of hESCs to hepatocytes by priming hESCs towards definitive endoderm with activin A and sodium butyrate prior to further differentiation to hepatocytes with dimethyl sulfoxide, followed by maturation with hepatocyte growth factor and oncostatin M. We have demonstrated that differentiation of hESCs in this process recapitulates liver development in vivo: following initial differentiation, hESCs transiently express characteristic markers of the primitive streak mesendoderm before turning to the markers of the definitive endoderm; with further differentiation, expression of hepatocyte progenitor cell markers and mature hepatocyte markers emerged sequentially. Furthermore, we have provided evidence that the hESC-derived hepatocytes are able to carry out a range of hepatocyte functions: storage of glycogen, and generation and secretion of plasma proteins. More importantly, the hESC-derived hepatocytes express several members of cytochrome P450 isozymes, and these P450 isozymes are capable of converting the substrates to metabolites and respond to the chemical stimulation. Our results have provided evidence that hESCs can be differentiated efficiently in vitro to functional hepatocytes, which may be useful as an in vitro system for toxicity screening in drug discovery.

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.

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.

Side Population Cells in Developing Human Liver Are Primarily Haematopoietic Progenitor Cells

Side population (SP) cells have recently been identified in a number of tissues although their phenotype and functional abilities are poorly understood. Surface marker characterisation and functional assessment of developing liver SP cells might allow for their isolation and manipulation using clinically relevant techniques. It was hypothesised that SP cells are present early during human liver development and contribute to haematopoietic and epithelial lineage generation. Whilst the SP population remained positive for CD34 during the 1st and 2nd trimester, 1st trimester SP cells were more highly enriched for haematopoietic and epithelial progenitor activity than those from the 2nd trimester in vitro. Marker expression and functional similarities indicate that SP cells in developing human liver may share a temporal relationship with oval/progenitor cells, responsible for liver regeneration after massive or chronic hepatic injury. Furthermore, modification of SP integrin expression during development suggests a potential adaptive interaction with niche components such as fibronectin. Improved understanding of developing human liver SP cells will contribute to the generation of novel cell-based therapies for liver disease.

Progress and Future Challenges in Stem Cell-derived Liver Technologies

The emergence of regenerative medicine has led to significant advances in the identification and understanding of human stem cells and adult progenitor cells. Both cell populations exhibit plasticity and theoretically offer a potential source of somatic cells in large numbers. Such a resource has an important role to play in the understanding of human development, in modeling human disease and drug toxicity, and in the generation of somatic cells in large numbers for cell-based therapies. Presently, liver transplantation is the only effective treatment for end-stage liver disease. Although this procedure can be carried out with high levels of success, the routine transplant of livers is severely limited by organ donor availability. As a result, attention has focused on the ability to restore liver mass and function by alternative approaches ranging from the bioartificial device to transplantation of human hepatocytes. In this review we will focus on the generation of human hepatic endoderm from different stem/progenitor cell populations with a view to its utility in regenerative medicine.

Third-degree Heart Block Associated with Bupivacaine Infusion Following Total Knee Arthroplasty. A Case Report

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.

Three-dimensional Culture of Human Embryonic Stem Cell Derived Hepatic Endoderm and Its Role in Bioartificial Liver Construction

The liver carries out a range of functions essential for bodily homeostasis. The impairment of liver functions has serious implications and is responsible for high rates of patient morbidity and mortality. Presently, liver transplantation remains the only effective treatment, but donor availability is a major limitation. Therefore, artificial and bioartificial liver devices have been developed to bridge patients to liver transplantation. Existing support devices improve hepatic encephalopathy to a certain extent; however their usage is associated with side effects. The major hindrance in the development of bioartificial liver devices and cellular therapies is the limited availability of human hepatocytes. Moreover, primary hepatocytes are difficult to maintain and lose hepatic identity and function over time even with sophisticated tissue culture media. To overcome this limitation, renewable cell sources are being explored. Human embryonic stem cells are one such cellular resource and have been shown to generate a reliable and reproducible supply of human hepatic endoderm. Therefore, the use of human embryonic stem cell-derived hepatic endoderm in combination with tissue engineering has the potential to pave the way for the development of novel bioartificial liver devices and predictive drug toxicity assays.

Portal Venous Endothelium in Developing Human Liver Contains Haematopoietic and Epithelial Progenitor Cells

Future treatments for chronic liver disease are likely to involve manipulation of liver progenitor cells (LPCs). In the human, data characterising the regenerative response is limited and the origin of adult LPCs is unknown. However, these remain critical factors in the design of cell-based liver therapies. The developing human liver provides an ideal model to study cell lineage derivation from progenitors and to understand how foetal haematopoiesis and liver development might explain the nature of the adult LPC population. In 1st trimester human liver, portal venous endothelium (PVE) expressed adult LPC markers and markers of haematopoietic progenitor cells (HPCs) shared with haemogenic endothelium found in the embryonic dorsal aorta. Sorted PVE cells were able to generate hepatoblast-like cells co-expressing CK18 and CK19 in addition to Dlk/pref-1, E-cadherin, albumin and fibrinogen in vitro. Furthermore, PVE cells could initiate haematopoiesis. These data suggest that PVE shares phenotypical and functional similarities both with adult LPCs and embryonic haemogenic endothelium. This indicates that a temporal relationship might exist between progenitor cells in foetal liver development and adult liver regeneration, which may involve progeny of PVE.

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.

Role of Stem-cell-derived Hepatic Endoderm in Human Drug Discovery

Accurate prediction of human drug toxicity is a vital part of the drug discovery process. However, the safety evaluation process is hindered by the availability and quality of primary human liver models with which to study drug toxicity. In an attempt to overcome this limitation, research has focused on deriving human hepatocytes from a number of sources, including progenitors from fetal and adult liver, human cell lines derived from liver tumours, immortalized human hepatocytes and pluripotent stem cells. The major hurdles in developing scalable and high-fidelity human hepatocytes from hepatic cell lines and fetal and adult progenitors have been limited organ availability, homogeneous cell purification, short-term cell culture, and the rapid loss of hepatocyte phenotype and function in culture. Therefore it has been necessary to find alternative sources of human hepatocytes which circumvent these issues. The research in our group has focused on generating human hepatic endoderm from the scalable pluripotent stem cell populations, human embryonic stem cells and induced pluripotent stem cells. We have developed efficient and scalable models of human hepatocyte differentiation from these cell populations. Moreover, stem-cell-derived hepatic endoderm displays many of the functional attributes of primary human hepatocytes. Our research is now focused on developing defined culture systems and improving cell culture microenvironments in order to improve our understanding of the mechanisms regulating human liver development. This will in turn facilitate the generation of broad-range functioning hepatic endoderm in vitro. By taking these approaches, we believe that it will be possible to improve the predictive nature of our in vitro models, revolutionizing the manner in which industry measures human drug toxicity and having an impact on drug attrition.

Cadaveric Hepatocytes Repopulate Diseased Livers: Life After Death

Pluripotent Stem Cell Derived Hepatocyte Like Cells and Their Potential in Toxicity Screening

Despite considerable progress in modelling human liver toxicity, the requirement still exists for efficient, predictive and cost effective in vitro models to reduce attrition during drug development. Thousands of compounds fail in this process, with hepatotoxicity being one of the significant causes of failure. The cost of clinical studies is substantial, therefore it is essential that toxicological screening is performed early on in the drug development process. Human hepatocytes represent the gold standard model for evaluating drug toxicity, but are a limited resource. Current alternative models are based on immortalised cell lines and animal tissue, but these are limited by poor function, exhibit species variability and show instability in culture. Pluripotent stem cells are an attractive alternative as they are capable of self-renewal and differentiation to all three germ layers, and thereby represent a potentially inexhaustible source of somatic cells. The differentiation of human embryonic stem cells and induced pluripotent stem cells to functional hepatocyte like cells has recently been reported. Further development of this technology could lead to the scalable production of hepatocyte like cells for liver toxicity screening and clinical therapies. Additionally, induced pluripotent stem cell derived hepatocyte like cells may permit in vitro modelling of gene polymorphisms and genetic diseases.

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 Complexities of Engineering Human Stem Cell-derived Therapeutics

Persistence of Functional Hepatocyte-like Cells in Immune-compromised Mice

Human embryonic stem cells (hESCs) can be efficiently differentiated to hepatocyte-like cells (HLCs) in vitro and demonstrate many of the functions and gene expression found in the adult liver.

Unbiased Screening of Polymer Libraries to Define Novel Substrates for Functional Hepatocytes with Inducible Drug Metabolism

Maintaining stable differentiated somatic cell function in culture is essential to a range of biological endeavors. However, current technologies, employing, for example, primary hepatic cell culture (essential to the development of a bio-artificial liver and improved drug and toxicology testing), are limited by supply, expense, and functional instability even on biological cell culture substrata. As such, novel biologically active substrates manufacturable to GMP standards have the potential to improve cell culture-based assay applications. Currently hepatic endoderm (HE) generated from pluripotent stem cells is a genotypically diverse, cheap, and stable source of "hepatocytes"; however, HE routine applications are limited due to phenotypic instability in culture. Therefore a manufacturable subcellular matrix capable of supporting long-term differentiated cell function would represent a step forward in developing scalable and phenotypically stable hESC-derived hepatocytes. Adopting an unbiased approach we screened polymer microarrays and identified a polyurethane matrix which promoted HE viability, hepatocellular gene expression, drug-inducible metabolism, and function. Moreover, the polyurethane supported, when coated on a clinically approved bio-artificial liver matrix, long-term hepatocyte function and growth. In conclusion, our data suggest that an unbiased screening approach can identify cell culture substrate(s) that enhance the phenotypic stability of primary and stem cell-derived cell resources.

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.

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