A study to improve the differentiation of human embryonic stem cells to functional hepatocytes

Abstract

Human embryonic stem cells (hESCs) possess 2 unique properties (1) pluripotency and (2) self-renewal, and therefore, hold great promise for biomedical application and regenerative medicine. In vitro differentiation of hESCs is a vital tool to generate unlimited human hepatocytes. To date, several multi-step protocols have been established to generate hepatocyte-like cells from undifferentiated hESCs via definitive endoderm (DE) formation. However, hESC derived-hepatocytes in these systems exhibit some immature characteristics, thus it remains a challenge on how to further improve hepatic differentiation. In addition, the molecular mechanisms regulating the differentiation are still ambiguous, making in vitro differentiation a difficult task. The ultimate aim of this project is to improve the differentiation of hESCs to functional hepatocytes. In this thesis, the work includes two main parts: (1) modulating signalling pathways to explore molecular mechanisms controlling DE differentiation; (2) developing a 3-dimensional (3D) culture system to improve the functionality of hESC-derived hepatocytes. DE formation is a critical step for the production of hepatocytes. In the first part of this thesis, I showed that suppression of PI3K signalling using the LY 294002 inhibitor (LY) during hESC differentiation significantly improves Activin A (AA)-induced DE generation, which subsequently augments hepatocyte production. Further mechanistic interrogation of this phenomenon has revealed that dual treatment of hESCs with AALY enhances the Activin downstream signalling, Smad2/3 phosphorylation and their nuclear translocation with Smad4. Furthermore, dual treatment with AALY also affects the disruption of β-catenin/E-cadherin complexes, which cooperatively contribute to distinctive morphological changes that may signify the occurrence of EMT and hence improved specification of DE. These findings suggest that suppression of PI3K/Akt modulates both Nodal/Activin and β-catenin pathways, the two most important signalling involved in mesendoderm and DE cell fate specification, therefore improved DE differentiation of hESCs. Liver development in vivo is regulated by cell–cell contacts in a 3D environment and the absences of this may account for, at least partly, some of the immature features of hESC-derived hepatocytes. In the second part of my thesis, based on initial encouraging results obtained from HepG2 cells, I applied alginate based 3D culture system to hESCs after they are differentiated into DE cells and optimised culture conditions. The results confirmed that 3D culture microenvironment enhanced hepatic differentiation and functionality of hESC-derived hepatocytes in compared to the monolayer format. Collectively, this study has demonstrated a significant cornerstone in the strategies to improve hepatic differentiation of hESCs by addressing the molecular signalling and micro-niche cues that govern hepatocyte lineage commitment. Hence, this will pave the way for the use of these hepatocytes in future regenerative therapies and biomedical applications.EThOS - Electronic Theses Online ServiceGBUnited Kingdo