Surgery (St Vincent's) - Theses

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Subject: tissue engineering × Author: Yap, Kiryu Kee Loong ×

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    Bio-engineering vascularised liver organoids
    Yap, Kiryu Kee Loong ( 2022)
    Liver organoids are bioengineered constructs that recapitulate native liver tissue, and are used to study liver development, test drugs, and as replacement tissue that can be transplanted to treat liver disease. This thesis focusses on the specific application of liver organoids for use as a cell-based treatment for liver disease via organoid transplantation. It places a particular emphasis on the addition of vascularisation to liver organoids to enhance structure and function, and improve engraftment during in vivo transplantation. Initially, the effect of the addition of endothelial cells to create vascularised liver organoids was assessed using mouse cells. The addition of mouse liver sinusoidal endothelial cells (LSECs) to mouse liver progenitor cells (LPCs) resulted in a striking change in organoid morphology, with the development of hepatobiliary ductular structures and clusters of polygonal hepatocyte-like cells which did not appear when LPCs were cultured alone as organoids. Furthermore, in vitro hepatobiliary gene expression, hepatic synthetic functions (albumin and apolipoprotein E production) and organoid viability was significantly increased by the addition of LSECs. Upon transplantation into vascularised chambers established in Fah-/- Rag2-/- Il2rg-/- (FRG knockout) mice, LPC only organoids had almost zero survival at 2 weeks, whereas LPC/LSEC organoids developed robust hepatobiliary ductular structures with a 115-fold increase in HNF4a+ cells and 42-fold increase in Sox9+ cells. To translate the mouse findings into a humanised platform, human LPCs and LSECs and their human induced pluripotent stem cell (hiPSC)-derived counterparts were characterised. The hepatic differentiation of human primary adult LPCs and hiPSC derived LPCs into hepatocyte-like cells was confirmed based on cell morphology, marker expression, and function (albumin production), and transcriptomic profiling using bulk and single cell RNA sequencing. Concurrently, human primary adult LSECs were compared to hiPSC-derived endothelial cells(iECs). Although in vitro iECs had a generic endothelial phenotype very different to LSECs, when iECs were transplanted into mouse liver they underwent tissue specification to approximate LSECs, highlighting the importance of the liver microenvironment in this process. Subsequently, three types of vascularised human liver organoids were explored using primary human and hiPSC-derived cells. First, primary LPCs, LSECs and adipose-derived mesenchymal stem cells were combined in a human liver-derived extracellular matrix (ECM) hydrogel and seeded into bioabsorbable porous polyurethane scaffolds. Second, iECs were aggregated with hiPSC-derived hepatocyte (iHep) organoids to coat the surface of the iHep organoids. Third, single cell-type organoids were integrated to form a combination organoid created from hepatocyte, cholangiocyte, and vascular organoids. Of the three models, combining organoids of different cell types to create a combination organoid was deemed the best approach to derive complex liver organoids containing well-organised tissue structures such as polarised hepatocytes with bile canaliculi, bile ducts, and blood vessel networks. An overarching theme is that vascularisation is pivotal in the development of transplantable liver organoids. Adding endothelial cells promotes hepatobiliary differentiation, and pre-formed vasculature can significantly enhance the survival of transplanted liver organoids by hastening connection to the host’s blood supply. However, this is not easy to achieve and remains a challenge in the liver organoid field, and the production of well-vascularised liver organoids with sustained development of blood vessels over time in culture remains elusive. Nevertheless, the limitations and challenges identified in this thesis point towards future directions in addressing the issue of vascularisation. For clinical translation of liver organoid transplantation, hiPSC-derived cells are a more reliable source of personalised cells, and the ECM additive should be bio-synthetic and chemically-defined, rather than Matrigel. Ultimately, the results in this thesis support the exciting prospect of stem-cell derived liver organoids being used as a regenerative treatment for liver disease.