Liver disease is a growing clinical burden worldwide. International rates of obesity and the incidence of alcohol abuse in Europe contribute significantly to chronic liver disease. Approximately a quarter of the world's population is estimated to have non-alcoholic fatty liver disease, and alcohol abuse is the most common cause of liver failure in many European countries. At present, the only cure for chronic liver failure is liver transplantation. However, the mismatch between patient demand and donor supply is widening. Regenerative medicine and tissue engineering including cell therapy and lab-grown mini-livers offer an exciting alternative to liver transplantation. Currently, two significant limitations in these fields are the immaturity of lab-grown hepatocytes and mini-livers, and the sub-optimal biomaterials available for tissue engineering and cell delivery. For example, Matrigel is the most commonly used matrix in hepatocyte differentiation from pluripotent stem cells and liver organoid culture, even though Matrigel is prohibited from clinical use.

The work for this Doctor of Philosophy was aimed to address the limitations of current biomaterials. The primary objective was to explore proteins within the hepatocyte niche and develop a novel hydrogel that can replace Matrigel in the three-dimensional differentiation of liver tissue from pluripotent stem cells for liver tissue engineering. The secondary objective was to explore its potential for clinical translation in murine models of transplantation to help address the unmet clinical need.

To achieve these objectives, a high throughput imaging platform was first developed to identify critical extracellular matrix proteins involved in the maintenance of stem cell pluripotency and hepatocyte differentiation. Next, a selection of hydrogel-forming natural proteins was investigated for their suitability as a polymeric backbone to conjugate candidate proteins. Hydrogels comprising candidate-backbone conjugates were then characterised and evaluated for their ability to support the tri-lineage three-dimensional differentiation of pluripotent stems into hepatic, cardiac, and neural lineages. Finally, the biocompatibility and utility of this novel hydrogel were evaluated in murine models.

Results herein firstly show the high discriminatory ability of the developed high-throughput imaging platform. Subsequently, several Laminin sub-types that contributed to stem cell pluripotency and hepatocyte differentiation were successfully identified by screening the hepatocyte niche proteins. Next, the conjugation of selected laminins to serum-derived proteins led to the discovery of a novel albumin-based hydrogel with potential in bone engineering and a fibrin-based hydrogel (termed "Alphagel") with a strong potential for clinical translation. Alphagel was shown to support three-dimensional stem cell differentiation and, therefore, could be utilised in whole organ tissue engineering. Furthermore, it was demonstrated that Alphagel, being made of clinical-grade components, was biocompatible in vivo and could be loaded with liver-specific proteins (termed "Hepatogel") to achieve better cellular phenotypes.

In conclusion, this work has demonstrated that Alphagel is a viable alternative to Matrigel for three-dimensional stem cell differentiation, tissue engineering, and cell or tissue transplantation. In addition, tissue-specific hydrogels like Hepatogel are likely to produce better cellular phenotypes than generic hydrogels like Matrigel. Therefore, future work will focus on developing tissue-specific hydrogels and investigating their utility in large animal studies and human disease modelling.