Sebastian Greenhough

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.

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.

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.

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.

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.

Differentiation and molecular profiling of human embryonic stem cell-derived corneal epithelial cells

It has been suggested that the isolation of scalable populations of limbal stem cells may lead to radical changes in ocular therapy. In particular, the derivation and transplantation of corneal stem cells from these populations may result in therapies providing clinical normality of the diseased or damaged cornea. Although feasible in theory, the lack of donor material in sufficient quantity and quality currently limits such a strategy. A potential scalable source of corneal cells could be derived from pluripotent stem cells (PSCs). We developed an in vitro and serum-free corneal differentiation model which displays significant promise. Our stepwise differentiation model was designed with reference to development and gave rise to cells which displayed similarities to epithelial progenitor cells which can be specified to cells displaying a corneal epithelial phenotype. We believe our approach is novel, provides a robust model of human development and in the future, may facilitate the generation of corneal epithelial cells that are suitable for clinical use. Additionally, we demonstrate that following continued cell culture, stem cell-derived corneal epithelial cells undergo transdifferentiation and exhibit squamous metaplasia and therefore, also offer an in vitro model of disease.

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.

Development of an Embryoid Body–Based Screening Strategy for Assessing the Hepatocyte Differentiation Potential of Human Embryonic Stem Cells Following Single-Cell Dissociation

We have devised an embryoid body-based screening method for the selection of human embryonic stem cell (hESC) lines capable of forming functional hepatocyte-like cells (HLCs) after single-cell dissociation. The screening method highlighted one cell line from a panel of five that produced albumin-positive cells during embryoid body (EB) formation. Cell lines that did not produce albumin-positive cells during EB formation were shown to respond less well to directed differentiation following single-cell replating. Additionally, the seeding density of the pluripotent populations prior to differentiation was shown to exert a significant effect on the hepatic function of the final population of cells. In summary, we have developed a simple procedure that facilitates the identification of human hESC lines that tolerate single-cell replating and are capable of differentiating to HLCs. Although the hepatic function of cells produced by this method is ∼10-fold lower than our current gold standard stem cell-derived models, we believe that these findings represent an incremental step toward producing HLCs at scale.

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.