Introduction Liver cells hepatocytes metabolise

Introduction Liver antioxidants (hepatocytes) metabolise most compounds and therefore can be used to predict liver metabolism and drug toxicity of a drug/compound. However most metabolic models are typically based on rodent liver (for reasons of supply and uniformity) and therefore only approximately predict human drug metabolism (Guillouzo, 1998; Maurel, 1996). As a result many drug trials have had to be terminated because such systems simply failed to predict human drug toxicity. The alternatives to rodent hepatocytes are cryopreserved or freshly isolated primary human hepatocytes. However, their widespread use is limited by supply, expense, variability in quality, and the diminishing function and viability following isolation and cryopreservation. However, despite these limitations they continue to serve as the industrial gold standard. Thus, there is an unmet need for the development of more accurate and predictive toxicity models. hESCs represent a pluripotent cell population able to generate all primary cell types in vitro (Thomson et al., 1998; Reubinoff et al., 2000). The ability to derive unlimited amounts of human HE from pluripotent stem cells constitutes an attractive and scalable resource which holds great potential. The deployment of this resource in the drug discovery process has the potential to streamline and standardize the predictive process and therefore impacting on the costs of drug attrition. Although very promising (Hay et al., 2008; Basma et al., 2009; Agarwal et al., 2008; Baharvand et al., 2006; Duan et al., 2007; Lavon et al., 2004; Touboul et al., 2010; Hannoun et al., 2010; Greenhough et al., 2010; Payne et al., 2011), hESC-derived HE, like primary human hepatocytes, also demonstrate reduced long-term viability and function in prolonged tissue culture, meaning that the advantages gained in uniformity, supply, integrity, and specificity have to date been offset by inflexibility necessitated by short-term culture models. Using an unbiased approach and capitalising on our recent demonstration that hESCs can be differentiated to form HE in vitro (Hay et al., 2008), we employed the polymer microarray screening approach (Pernagallo et al., 2009; Tourniaire et al., 2006; Thaburet et al., 2004) to discover novel extracellular substrate(s). We identified a simple polyurethane which not only provides long-term hepatic support, but does so in a manner in which the hepatocytes are fully functional and exhibit superior drug inducibility to freshly isolated primary human hepatocytes (FPHH). The data provide proof of concept that a novel and unbiased approach offers a simple and cost-effective means of identifying alternatives to complex xenobiotics. Moreover, such polymers provide a scalable resource to support differentiated cell function in research, drug screening, and toxicology and in the longer term, extracorporeal devices.
Results
Discussion In an attempt to improve the quality of hESC-derived HE we screened polymer libraries for novel and bioactive substrata. Following polymer library screening we identified 6 polymers which supported hepatic attachment and identity in vitro (Fig. 1). In order to study hepatic function in further detail the hit polymers were screened for the expression of a panel of soluble export proteins produced by the liver. From these studies polymer 134 was identified as the most effective cellular support (Fig. 2). In addition to changes in levels of HE function we also noted gross changes in cellular morphology with hESC HE replated on 134, exhibiting clear hepatic morphology and canaliculi (Fig. 4). In line with changes in cellular morphology we also observed major differences in HE signaling networks, consistent with the cells remaining hepatic and firmly attached to the substrate (Fig. 4). In addition, HE maintained on polymer 134 displayed similar CYP3A function to cryopreserved PHHs which was superior to the all other ECMs tested (Fig. 4, Supplementary Fig. 1). This being said, both hESC-HE and cryopreserved PHHs displayed reduced basal function (~10- to 15-fold) when compared to freshly isolated PHHs. Although basal function was reduced in vitro, dose-dependent drug induction of CYP3A drug metabolism was superior in hESC-derived HE compared to freshly isolated PHHs (Fig. 6 and Supplementary Fig. 4), indicating that hESC-derived HE was a more sensitive, and therefore predictive, model of human liver function in vitro.