PhD Studentship: Development of a Human Hepatocyte Culture System to Simultaneously Measure Drug-Induced Liver Injury and Determine the Hepatobiliary Elimination Transporter(s)

Supervisor

Prof Kenneth Linton, k.j.linton@qmul.ac.uk, Professor of Protein Biochemistry, Queen Mary University of London

Project Details

The Problem

Inhibition of bile flow by off target effects of therapeutic drugs is a significant clinical problem and such drug-induced liver injury (DILI) is the major cause of failure of drugs in human trials.

Bile flow and the identification of the drug elimination transporters in the liver are not routinely measured in hepatic cultures in the pharmaceutical industry. Simple inhibition of the Bile Salt Export Pump in vitro combined with toxicology tests in rodents are the accepted standards expected by the Medicines Agencies.

Our Aim

We aim to improve preclinical testing and remove the need for animal models in hepatotoxicity tests. To achieve this goal we will engineer (and test) a series of human hepatic cell lines that recapitulate all functions of the liver, including bile formation and flow, but lack one of the several drug efflux pumps normally present on the canalicular membrane. This will allow us to understand the drivers of more complex liver injury, beyond simple BSEP inhibition, and by developing cell lines that are deficient in the drug efflux pumps we will be able to identify the drug (and drug metabolite) elimination transporters, simultaneously.

The Plan of Action

In collaboration with AstraZeneca we have developed a protocol to differentiate human hepatocytes from induced pluripotent stem cells (iPSCs)[1]. These induced-hepatocytes (iHEPs) are grown in multiwell plates between two layers of extracellular matrix (ECM) to mimic the minimal functional unit in the liver: the hepatic plate (Figure 1a). The iHEPs are multipolar (a unique property of hepatocytes in the liver) and form compartments between cells (canaliculi) into which bile is secreted. These are readily visualised using a fluorescent compound which is transported by the drug efflux pump MRP2 (Figure 1a). Gene and protein expression data demonstrate that these cells are fully functional for drug metabolism. Bile flow from the liver is the main faecal elimination pathway for many therapeutic drugs and drug metabolites. By removing calcium, which releases the tight junctions that delineate the canaliculi, we can recover the bile. The key transporters that secrete drugs and drug metabolites across the canalicular membrane are the efflux pumps ABCB1, ABCG2, MRP2 and MATE1 but the importance of each for individual compounds is often unknown. By starting with iPSCs we are able to systematically knock out any gene of interest by genome editing and engineer cell lines to investigate the importance of each transporter.

Objectives

We will refine and exploit our iHEP culture system to develop a robust and sensitive test bed for drug toxicology and pharmacokinetic studies.

This will be achieved in four steps:

1. We will refine our protocol to replace the Matrigel (a product derived from mice) as the ECM. A synthetic matrix will be developed to eliminate potential contamination by rodent cytokines and develop a fully animal-free test protocol.
2. The iHEP culture system will be developed and scaled to optimise recovery of the canalicular bile for mass spectrometry.
3. CRISPR-Cas9 will be used to generate knock-out cell lines, each lacking one of the drug efflux pumps.
4. Validation with test drugs will establish (a) the elimination route for the drugs/metabolites by comparing flux through the cell lines developed in (3) with the wild-type hepatocytes (b) Measurement of key factors of the adaptive and deteriorative responses will simultaneously measure DILI.

Outcomes

• Identification of new off-targets that lead to DILI.
• Insight into the fundamental changes that lead to adverse outcomes.
• Replacement of animal models for drug testing with physiologically-relevant human cells that are more sensitive for detection of DILI.
• Lab technique acquisition

The project will develop independent, creative, thinking in an academic and industry setting. In addition to developing a breadth of transferable skills through the LIDo programme and doctoral college of QMUL, you will learn a diverse range of laboratory techniques, including genome editing, pluripotent stem cell biology, fluorescence microscopy, mass spectrometry, pharm