Next-generation in vitro liver models through stem cell-derived organoids and bioengineering

Liver research has historically heavily relied on animal models and primary human cells, as liver function could only be inadequately recapitulated in in vitro models. The advent of liver organoid technologies offered enormous potential to support liver research. However, in contrast to other adult-stem cell derived organoids, such as those of the intestine or lung, the formation of liver organoids is governed by a cell-type specific regenerative response and not by the expansion of multipotent progenitor cells. Therefore, cholangiocyte and hepatocyte organoids were developed separately, with limited capacity for giving rise to one another. Cholangiocyte organoids have enabled the study of basic bile duct pathophysiology. However, their closed, cystic morphology poses significant drawbacks, such as limited culture period, inaccessible lumen, and lack of in vivo-like cellular organization. To address these challenges, I first adapted an organ-on-chip technology to create a bioengineered bile duct model with organoids. These tubular organoids could be stably cultured for at least 45 days and exhibited key physiological characteristics, such as transport activity, primary cilia formation and a protective glycocalyx. In addition, the tubular organoids were susceptible to physical and chemical damage, an aspect that cannot be replicated with such precision in conventional organoids. To achieve greater tissue-level complexity, a perfusable branching network was engineered and cellular diversity was augmented by co-culturing with vascular endothelial cells and fibroblasts. Despite significant efforts, long-term expansion of human hepatocyte organoids has been largely limited to those derived from fetal tissue, which is not a readily accessible cell source. Leveraging the potential of human pluripotent stem cells, I successfully developed a new protocol to generate both hepatocyte and cholangiocyte organoids from bipotent liver progenitors termed hepatoblasts. These organoids exhibited robust expansion capacity and stability over at least 16 passages, maintaining both proliferative capacity and lineage-specific characteristics. Single-cell transcriptome analyses confirmed the efficient acquisition of maturing cellular identities in both organoid types, with a subpopulation of hepatocyte organoids exhibiting stem/progenitor-like characteristics akin to fetal tissue-derived counterparts. Furthermore, transcriptional profiling of hepatoblasts and organoids revealed their strong resemblance to the respective in vivo liver developmental stages and, most importantly, their comparability with organoids derived from primary cells. By harnessing the power of stem cell biology, organoids and bioengineering, this work provides a new set of tools to push the boundaries of our understanding of the complex mechanisms underlying liver health and disease.