Abstract
dc:description.abstractUnderstanding human development, specifically the initiation of human organogenesis, remains a major challenge not only in the field of developmental biology but also to understanding regenerative processes and developmental disorders. However, studying the initial stages of human organ development remains out of reach due to technical and ethical limitations. In the context of the hepatobiliary system, this challenge is particularly apparent. Indeed, current models of hepatobiliary development remain extremely rudimentary, lack the cellular complexity of natural tissue and do not produce organ progenitors with full developmental potential. To address these major questions, I have developed a system which produces a complex of organ progenitors from human pluripotent stem cells which can self-organise into hepatobiliary bud (HepBud) organoids when grown in 3D. This method forms a heterogenous differentiation culture of foregut derivatives which regionalise to form pancreatic, hepatic and biliary progenitors combined with cardiac and endothelial cells. After 3D seeding, these populations self-organise into complex budding structures which recapitulates the structural complexity of the developing liver bud. This includes a ‘bud’ containing hepatoblast progenitors (ALB+AFP+HNF4A+), and a ‘trunk’ containing hepato- pancreato-biliary progenitors (KRT19+CDX2+PDX1+HNF1B+) and extra-hepatic cholangiocytes (ACE2+MUC13+). By performing extensive snRNAseq analyses across HepBud formation, I was able to validate that hepatic specification in HepBuds follows a natural developmental path forming hepatoblast which strongly resemble 5/6pcw human foetal hepatoblasts. Moreover, by examining cell:cell interactions I found that early 2D HepBud cultures mimic the niche of cellular interactions of the developing liver bud, implicating cellular complexity as the key to generating bonafide hepatoblast progenitors in vitro. I go on to demonstrate the developmental potential of HepBuds by deriving homogenous self-renewing hepatoblast organoids (iHBOs). These iHBOs display a capacity to differentiate into intra-hepatic ductal plate cholangiocytes, hepatocytes and could even be induced to form functional intra-hepatic cholangiocyte organoids (iCLOs). Critically, iHBOs and iHBO-derived hepatocytes and cholangiocytes share many key functional and transcriptional features with their in vivo counterparts. Finally, iHBOs also display a capacity to differentiate and mature in vivo following intra-splenic transplantation into mice under a model of acute liver injury. Taken together, these results show that our model of liver bud development provides a new platform not only to study hepatobiliary lineage specification in vitro but also to produce cell types with a clinical interest.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2025
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Grey-Wilson, Lottie
- Advisors dc:contributor.advisor
-
- Vallier, Ludovic
- Sinha, Sanjay
Subjects
dc:subject × 7Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.121169
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/389132