Many hepatic cell lines have been derived but they are all limited by the down-regulation or loss of a key function of the liver: bile synthesis.
In this project you will develop a novel transwell perfusion system for hepatocyte culture that maintains bile formation and flow. This will allow you to induce cholestasis and define the fundamental changes which drive progression of complex liver diseases. This project brings together experts in bile flow transporters (Prof Linton, Centre for Cell Biology, Blizard Institute, Queen Mary University of London), differentiation of induced pluripotent stem cells (iPSCs) into hepatocytes (Dr Rashid, Centre for Stem Cell and Regenerative Medicine, King’s College London) and a global pharmaceutical company (Dr Williams, Hepatic Safety group, AstraZeneca, Cambridge). You will work in all three groups to gain complementary expertise to address the project aims.
What is bile and why is it important?
Bile is a complex mixture of bile salts (BS), the membrane lipid phosphatidylcholine (PC), cholesterol, salts and waste products that is formed in the biliary canaliculi and stored in the gallbladder, prior to entering the gut. It is a key product of the liver and necessary to solubilise dietary fat and vitamins. Impaired bile flow (cholestasis) causes liver damage because the bile salts, which are strong detergents, accumulate. Patients with cholestasis present with acute or chronic liver disease, depending on the level of impairment in the bile flow transporters. The critical transporters driving bile flow from the hepatocyte, the BS export pump (BSEP) and the PC floppase (ABCB4) have been identified and characterised in our lab (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 transporter inhibition which are much more prevalent, cause chronic symptoms that predispose to the development of a spectrum of liver disease including gestational cholestasis, gallstone disease, biliary cholangitis, sclerosing cholangitis and hepatocellular cancer (reviewed in Nicolaou et al., J Path 2014).
The molecular changes associated with this cholestatic predisposition to secondary pathologies have not been described. Progress has been slow because the biliary canaliculi in vivo is inaccessible, which precludes sampling, and because hepatocytes cultured in vitro rapidly lose the ability to make bile (in systems where low-level bile formation is maintained, e.g. in 3D cultures, it is secreted into closed compartments within the spheroid, preventing accurate kinetic measurements).
What you will do Hepatocytes (iHEPs) will be induced to differentiate from iPSCs using technology developed in the Rashid lab (Rashid et al., JCI 2010; Yusa et al., Nature 2011). Mutations leading to the development of cholestasis (Andress et al., Hepatol 2014 and Cell Mol Life Sci 2017) will be introduced into the key transporters by CRISPR/Cas9, or cholestasis will be induced by inhibition with gestational hormones and/or cholestatic drugs. These cells will be cultured on a porous membrane in a transwell system, along with extracellular matrix, and other liver cell types to mimic liver tissue. Micro-fluidic channels will provide continuous laminar flow of nutrients and oxygen to the basal compartment, mimicking the portal vein, while harvesting bile from the apical compartment. Bile complexity and flow will be characterised before measurement of changes in gene expression and cytokine release into the apical and basal chambers following induction of cholestatic conditions.
The development of a novel hepatocyte culture method that mimics liver physiology, including the synthesis and flow of bile, will allow you to:
- define the initial responses of hepatocytes following induction of cholestasis
- identify new markers of disease
- identify damage-associated molecular patterns that predict adverse outcomes and development of more complex liver disorders
Developing fundamental understanding of the disease pathology will have major basic science and translational implications, enabling the development of new tools for early diagnosis and intervention in liver disease.
This challenging project offers great scope for innovation and creativity and provides a platform to learn several cutting-edge techniques including: perfused tissue culture technology; CRISPR/Cas9 editing; bile and cytokine metabolomics; RNA seq; QRT-PCR; Western blot analysis.
Applications Applications must be complete, including two references, by 19th January 2018 at 5pm
Fully funded place including home (UK) tuition fees and a tax-free stipend in the region of £16,553. Students from the EU are welcome to submit an application for funding, any offers will be subject to BBSRC approval and criteria.
Groen A, Romero MR, Kunne C, Dixon PH, Wooding C, Williamson C, Seppen J, van den Oever K, Ho-Mok KS, Paulusma CC, Linton KJ and Oude Elferink RPJ. The flippase ATP8B1 counteracts the deleterious function of floppase ABCB4 to maintain hepatocanalicular membrane integrity. Gastroenterol. (2011) 141, 1927-37.
Byrne JA, Strautnieks SS, Mieli-Vergani G, Higgins CF, Linton KJ and Thompson RJ. The human bile salt export pump: characterization of substrate specificity and identification of inhibitors. Gastroenterology (2002) 123, 1649-58.
Nicolaou M, Andress EJ, Zolnerciks JK, Dixon PH, Williamson C, Linton KJ. Canalicular ABC transporters and liver disease. J Pathol. (2012) 226, 300-15.
Andress EJ, Nicolaou M, McGeoghan F, Linton KJ. ABCB4 missense mutations D243A, K435T, G535D, I490T, R545C, and S978P significantly impair the lipid floppase and likely predispose to secondary pathologies in the human population. Cell Mol Life Sci (2017) 74: 2513-24.
Andress EJ, Nicolaou M, Romero MR, Naik S, Dixon PH, Williamson C and Linton KJ. Molecular mechanistic explanation for the spectrum of cholestatic disease caused by the S320F variant of ABCB4. Hepatology (2014) 59: 1921-31.
Rashid ST, Corbineau S, Hannan N, Marciniak SJ, Miranda E, Alexander G, Huang-Doran I, Griffin J, Ahrlund-Richter L, Skepper J, Semple R, Weber A, Lomas DA, Vallier L. Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. J Clin Invest (2010) 120(9):3127-36.
Yusa K, Rashid ST, Strick-Marchand H, Varela I, Liu PQ, Paschon DE, Miranda E, Ordóñez A, Hannan NR, Rouhani FJ, Darche S, Alexander G, Marciniak SJ, Fusaki N, Hasegawa M, Holmes MC, Di Santo JP, Lomas DA, Bradley A, Vallier L. Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells. Nature (2011) 478(7369):391-4.