The role of the mechanical microenvironment in regulation of cholangiocyte specification and tubulogenesis

Abstract

Emerging evidence highlights the influence of the extracellular matrix (ECM) on cell fate and functionality associated with ageing, development and disease (Segel et al. 2019; Bansaccal et al. 2023). Despite most studies emphasising ECM stiffness, recent insights suggest a cooperative role of ECM ligand composition and stiffness in driving biological function. However, a major constraint restricting previous studies is the inability to control ECM composition and stiffness in 3D. As a result, modelling the microenvironmental influence on 3D organ morphogenesis in vitro has been limited. In this dissertation, I built upon our previously established polyacrylamide hydrogels (Labouesse et al. 2021) alongside our 3D culture system (Mulas et al. 2020) to develop a tuneable 3D hydrogel, allowing for adjustment of ECM stiffness and composition. I then employed this system to study bile duct specification and morphogenesis in vitro using well-established differentiation protocols (Hannan et al. 2013b; Sampaziotis et al. 2017). Through carefully controlling the ECM, I observed that high densities of collagen type I serve as an essential signal for the developing bile ducts, overriding the influence of substrate stiffness. This was supported by in vivo evidence, as, although collagen I is absent from the main parenchyma of the human foetal liver, I found that high-density regions surround the developing bile ducts. Furthermore, I showed that inhibition of this cell-ECM interaction results in progenitors retaining high expression of hepatic markers during biliary epithelial cell differentiation, indicating an insufficient fate instruction. A process which I linked to enhanced activity of the mechanosensitive YAP/TAZ signalling cascade, a purported component of biliary differentiation. Finally, I demonstrated how collagen I-binding mechanisms link to bile duct morphogenesis through the development of 3D tubular bile ducts. Inhibition of this binding results in aberrant tube formation, emphasising collagen I’s role in both cholangiocyte fate specification and morphogenesis. Together, my findings reveal how cell-ECM interactions are a primary regulator of biliary epithelial cell fate specification and morphogenesis. This work, to my knowledge, is the first to demonstrate the essential influence of collagen type I on biliary epithelial cell fate choice and morphogenesis during liver development. Going forward, this will serve as a stepping stone towards a unified view of ECM composition and niche mechanics in cell fate determination and morphogenesis.