AbstractOrthotopic liver transplantation is the only effective treatment for end-stage liver diseases; however, it is associated with various drawbacks e.g., risk of rejection and lifelong immunosuppression impacting the quality of life. This has led to exploration of induced pluripotent stem cells (iPSCs) as sources of human hepatocytes. Various research groups have successfully derived hepatocytes from iPSCs and demonstrated their ability to support impaired liver function upon transplantation. Despite the continuous efforts within the field, differentiation of human iPSCs to cells with sufficiently equivalent function to primary hepatocytes to replace OLT has not been achieved to date.
It is widely accepted that the phenotypic expression and function of stem cells are regulated by their integrated response to microenvironmental factors which include chemical and mechanical cues. The nucleus has emerged as an important mechano-sensory organelle in the cell, with studies providing evidence that it can respond to topographical stimuli which can modulate the epigenetic landscape and influence expression of genes. The aim of this research is to develop a strategy to improve the functional phenotype of iPS-derived hepatocytes.
I developed a strategy that significantly improves the function of iPS-derived hepatocytes by combining established hepatic chemical induction protocols with topographical stimulation. First, I developed and optimised a microgroove platform for topographical stimulation of iPSCs. Next, I defined the effects of direct topographical stimulation of iPSCs on their differentiation and function into hepatocytes. Topographical constriction of iPSCs improved the phenotype of derived hepatocytes by significantly increasing the albumin production rate and the drug metabolism function quantified by CYP3A4 expression. The data indicates a possible role of nuclear mechanosensing, as it shows the most improved phenotype in cells with their nuclei confined while engaging in cell-cell contact. While the precise mechanisms behind this microgroove-induced improvement in function is still to be determined, the data in this thesis highlights a possible epigenetic mechanism behind the microgroove-induced functional improvement. Additionally, the observed effect of topographical stimulation is transient, thus continuous stimulation would be required to maintain the improved functional phenotype should this strategy be employed in vivo. I optimized the generation of biodegradable polycaprolactone microgrooves, providing a foundation for the implantation of iPS- derived hepatocytes within microgrooves for further in vivo exploration. Further studies are required before this strategy may be considered in the clinic; however, it can be used as a method to generate iPS-hepatocytes with an improved functional phenotype for research applications.
Overall, the results presented in this thesis provide the cornerstone for future research into topographical stimulation for the generation of functional hepatocytes with enhanced functions.
|Date of Award
|1 Dec 2022
|Ciro Chiappini (Supervisor) & Tamir Rashid (Supervisor)