Modelling bile formation and flow in vitro to understand normal healthy liver function and how damage-associated molecular changes predispose towards

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Bile, a complex mixture of bile salts (BS), phosphatidylcholine (PC), cholesterol, salts and waste products, is formed in the biliary canaliculi. Impaired bile flow (cholestasis) causes liver damage and patients present with acute or chronic disease. The key transporters driving bile flow from the hepatocyte, the BS export pump (BSEP) and the PC floppase (ABCB4), have been identified and characterised in the KJL and RJT labs (Groen et al Gastro 2011; Byrne et al., Gastro 2002). Null mutations in these transporters cause acute forms of progressive familial cholestasis that are fatal in childhood in the absence of liver transplant. Milder forms of transporter insufficiency (or inhibition), which are much more prevalent, predispose to development of a spectrum of liver disease including gestational cholestasis, gallstone disease, biliary cholangitis, sclerosing cholangitis and hepatocellular cancer (Nicolaou et al J Path 2014). The molecular changes associated with predisposition to secondary pathologies have not been described. Progress is slow because the biliary canaliculi in vivo is inaccessible precluding sampling and also because hepatocytes cultured in vitro rapidly lose the ability to make bile (where low level bile formation is maintained, e.g. in 3D cultures, it is secreted into closed compartments internal to the spheroid preventing accurate kinetic measurements).We will develop a novel transwell perfusion system for hepatocyte culture that maintains bile formation and flow (Fig 1) and this will allow us to induce cholestasis and define the fundamental changes that drive disease progression. Hepatocytes (iHEPs) will be derived from iPSCs using technology developed in the TR lab (Rashid et al., JCI 2010, Nature 2011). Homozygous ABCB4-S320F mutations will be introduced in the iPSCs prior to differentiation. ABCB4-S320F, unusually, causes a spectrum of liver pathologies (Andress et al., Hepatol 2014). Cholestasis will be induced by inhibition of wild type (WT) or ABCB4-S320F by gestational hormones and/or cholestatic drugs as necessary. Bile complexity and flow will be characterised before measurement of changes in gene expression and cytokine release into the apical and basal chambers.