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.
- cell culture
- drug discovery
- embryonic stem cell
- hepatic endoderm
- induced pluripotent stem cell
Previous approaches and limitations
Hepatocytes represent the main cell type of the liver parenchyma (~70%), performing a variety of important endocrine and exocrine functions essential for bodily homoeostasis. Therefore the use of ex vivo adult PHHs (primary human hepatocytes) is an obvious and desirable option for both basic liver research and the development and safety evaluation of new drugs. However, this approach has been severely hampered by the difficulties associated with organ availability, hepatocyte isolation, the use of diseased tissue and the maintenance of PHH phenotype and critical function in vitro.
In an attempt to overcome these difficulties, a number of strategies have been employed, from the purification of human liver stem cell populations to the generation of human hepatic cell lines and the use of surrogate animal models. A number of studies have proposed the use of hepatic progenitor cells found in the early fetal liver bud (hepatoblasts) and adult liver stem cells (oval cells) for the generation of primary hepatocytes. These hepatic progenitor cells have the potential to give rise to both hepatocytes and cholangiocytes [1,2]. Although hepatic progenitor cells provide a valuable source of liver cells, they have serious limitations, including difficulties with their homogeneous isolation and in vitro maintenance, hindering their large-scale expansion and application . Human hepatic cell lines have been isolated ex vivo from hepatic tumours or generated by immortalization. Despite these attempts, the derivative cell lines still display varying liver function in vitro, contain poor levels and inducibility of key metabolizing enzymes, and do not possess normal or stable karyotypes. Rodent hepatocytes and in vivo models of liver toxicity have also been employed with limited success. A major problem with this approach is the extrapolation of data to humans, which is complicated by species-specific variations in response to drugs and idiosyncratic drug reactions within the same species. Although major progress has been made in the field, there is a clear imperative that new predictive models are required in order to improve safety evaluation and reduce the spiralling costs associated with drug attrition.
hESCs [human ESCs (embryonic stem cells)] and iPSCs [induced PSCs (pluripotent stem cells)] offer an inexhaustible cellular resource
Owing to the problems discussed above, attentions have focused on the potential that other cell populations may have to offer in this field. Both hESCs and more recently iPSCs have been isolated. hESCs are derived from the inner cell mass of blastocyst stage embryos and are highly primitive cells which demonstrate self-renewal and pluripotency [4,5]. iPSCs exhibit similar characteristics to those of hESCs and are generated by the forced expression of key transcription factors in adult somatic cells [6,7]. Key attributes of both stem cell populations are that they self-renew while retaining their developmental potency. This means that, in theory, these cell types have the ability to create an inexhaustible supply of primary human cell types such as human HE (hepatic endoderm) (for a review, see ). Moreover, the creation of iPSCs from human somatic cell types has circumvented the controversial issues associated with the use of human embryos for hESCs. Therefore hESCs and iPSCs represent a unique opportunity for improving cell-based applications for drug discovery, predictive toxicology testing and clinical therapies, which will have an enormous benefit for human health (for a review, see ).
We and several other groups pioneered the directed differentiation of HE from hESCs [10–15]. More recently, through reference to human development, we generated a highly efficient model of human HE which displays mature mitochondrial structure and many of the functions associated with adult PHHs . Since then, we have translated our knowledge in HE generation from hESCs to human iPSC lines, representative of both sexes and different ethnic origins . This has provided a proof-of-concept that the derivation of iPSCs from individuals with polymorphic or ethnic variations is directly applicable to the construction of cell-based models and provides the pharmaceutical industry with a unique opportunity. Additionally, the identification and reprogramming of somatic cells, coupled with direct differentiation to a specific lineage, represents a powerful resource with which to study human liver development and disease in tissue culture.
Defining in vitro culture conditions and producing quality assured technology
In recent years, a variety of approaches have been developed for the differentiation of hESCs into functional HE. In vitro culture models employed both 3D (three-dimensional) multicellular aggregate and 2D (two-dimensional) direct differentiation strategies supplying the appropriate developmental factors in a stage-wise fashion. Initially, murine ESCs and hESCs were differentiated in suspension cultures in which ESC aggregation resulted in the formation of EBs (embryoid bodies). These multicellular aggregates spontaneously differentiate into cell types representative of all three germ layers and can be replated on adherent matrices where, in the presence of growth factors and hormones, EB outgrowths exhibit HE differentiation [18–21]. However, despite EB formation providing a permissive microenvironment for HE differentiation, it is a spontaneous and stochastic process which occurs with limited efficiency, resulting in a mixed cell population of all three germ layers. More recently, impressive experiments by Basma et al.  demonstrated improved differentiation efficiencies and purified HE cell populations which exhibited hepatic function comparable with PHHs. Although these studies offer promise, the ability to scale-up 3D technologies at present is limited. Therefore attention has also focused on generating directed and 2D HE models from hESCs. Using a directed approach, we and others have generated efficient levels of HE displaying human hepatocyte function [10–16]. Most recently, we developed an efficient differentiation model employing activin A and Wnt3a signalling components which is scalable and applicable to both hESCs and iPSCs [16,17]. The challenge to scale-up these technologies in a defined and quality assured manner still remains and is an ongoing concern in many laboratories, including our own.
At present, most research laboratories maintain hESC and iPSC lines in tissue culture environments with undefined and xenobiotic components. This has limited the ability to derive large amounts of human HE from hESCs and iPSCs in a cost-effective manner. Defined cell culture technology and quality assurance will permit the production of HE in sufficient quantity for industrial and clinical applications. Indeed, the first steps in this process have begun with the commercial production of serum-free culture medium and other defined reagents which have proven successful in stem cell maintenance and efficient directed differentiation to HE in our hands. As culture definition gains momentum, it is imperative that those in the stem cell field agree on the standard operating procedures for reliable maintenance and differentiation of PSC populations.
Cell culture microenvironment influences HE function and viability
In addition to physiological differentiation of PSCs, it is also crucial to generate the correct cell culture microenvironment. The cell niche is naturally 3D and its biochemistry and topology strongly affect the differentiation and maturation process . Liver development in vivo occurs in three dimensions, yet cost-effective and scalable culture in vitro is best achieved in a 2D environment. The use of 3D modelling systems may offer insights into cellular interactions and physiology which could be mimicked in 2D in vitro models, improving stability of hepatic phenotype and function. That said, we must also explore the creation of cost-effective and defined 3D hepatocyte models. This is essential, as certain liver functions will only be active in three dimensions and, in addition to improving HE function, these studies will play an essential role in humanized bioartificial design and construction .
Although hESC- and iPSC-derived hepatocytes demonstrate a major headway in the field of human liver biology, other key issues need to be addressed. Stem-cell-derived hepatocytes after prolonged culture de-differentiate and die in a manner similar to PHHs. Therefore we have employed novel polymer microarray technology to try to bypass the issues associated with culturing stem-cell-derived HE. Our data presented at the Biochemical Society meeting provide critical proof-of-concept that this novel and unbiased approach may provide a simple and cost-effective means of identifying alternatives to complex extracellular support matrices which contain xenobiotics. Moreover, we have identified a GMP (good manufacturing practice)-compliant and scalable resource which supports hepatic function in drug screening and extracorporeal device settings.
Role of PSC-derived HE in human drug discovery
Drug development is a lengthy and costly process. For each new drug that reaches the market, approx. 10000 compounds have been tested pre-clinically. These values highlight the importance of developing more predictive human toxicity models for primary drug screening. One of the principal procedures for studying the safety of drug compounds is to measure liver toxicity using cell- and animal-based models. Although the use of ex vivo adult hepatocytes, human cell lines and animals are highly valuable commodities for toxicology studies, they have their limitations as described above. As described previously, the application of human PSCs in drug toxicology studies has the potential to drastically improve the human drug development process. Furthermore, iPSCs have provided an efficient model with which to study SNPs (single nucleotide polymorphisms) known to influence the metabolism and clearance of drugs, for example the metabolism of warfarin . Thus the application of iPSC technology may prove an invaluable tool for modelling drug metabolism and furthering our understanding of human disease .
Recent progress in the efficient generation of high-fidelity HE from different PSC populations has provided an enormous opportunity to improve the way we measure human drug toxicity. Although we have taken a major step forward using stem cell technology, hESC- and iPSC-derived HE function is still not as broad as that of adult PHHs. Therefore ongoing research should focus on the elucidation of other developmental factors and tissue culture microenvironments that are essential in producing the optimal stem-cell-derived model(s).
D.C.H was supported by an Research Councils UK (RCUK) Fellowship; C.N.M. and S.G. were supported by the UK Stem Cell Foundation and Scottish Enterprise.
Revolutionizing Drug Discovery with Stem Cell Technology: A Biochemical Society Focused Meeting held at GlaxoSmithKline, Stevenage, U.K., 18–19 January 2010. Organized and Edited by Aaron Chuang (GlaxoSmithKline, U.K.), Katy Gearing (GlaxoSmithKline, U.K.) and Melanie Welham (Bath, U.K.).
Abbreviations: 2D, two-dimensional; 3D, three-dimensional; EB, embryoid body; ESC, embryonic stem cell; HE, hepatic endoderm; hESC, human ESC; PSC, pluripotent stem cell; iPSC, induced PSC; PHH, primary human hepatocyte
- © The Authors Journal compilation © 2010 Biochemical Society