Targeted Differentiation of Pluripotent Stem Cells to Hepatocytes

Abstract

Pluripotent stem cells (PSCs) possess the ability to differentiate to virtually any cell type whilst retaining the capacity to self-renew. There is an unmet need for an inexhaustible supply of hepatocytes to perform drug toxicity and metabolism screens on early stage drugs. PSCs therefore hold potential to generate mature hepatocytes for pharmaceutical testing. To realise this potential, there is a requirement to recapitulate hepatocyte specification in vitro. There are two main bottlenecks in generating metabolically relevant hepatocytes. The first is the specification of definitive endoderm (DE), where it is known that the TGFβ family member Nodal is the key driver in vivo. Currently the closely related TGFβ family member Activin A (AA) is universally used to mimic this process. However, it has been observed that AA results in off-target gene modulation that may be deleterious to the production of DE; from which the hepatic lineage arises. Preliminary data from the McKay group has shown that AA stimulates endogenous Nodal activity when applied to PSCs; leading to the hypothesis that Nodal is the true driver of DE specification in vitro. To address this avenue of investigation, tools to modulate Nodal signalling were assessed in different cell culture systems. Findings included the need to use an appropriate viral promotor to efficiently express lentiviral vectors in PSCs. The second major bottleneck concerns hepatic maturation where the signalling pathways and key regulators in vitro are not fully understood. Consequently, current derived hepatocyte-like cells (HLCs) show low functionality. Utilising Plasticell’s CombiCult® allowed thousands of growth factor and small molecule combinations to be screened to define efficient differentiation protocols. Protocols were derived containing novel differentiation factors that give rise to CYP450 inducible HLCs. Lentiviral reporters were then used to determine optimum concentrations of small molecules and to test ligands for the upregulation of hepatic master regulators. Findings gained from this thesis have contributed new insights into the differentiation of PSCs to hepatocytes.