Y29 - Stem cell-derived fusable assembloids to model human liver development

Introduction: : Chronic liver diseases are one of the leading causes of morbidity and mortality globally with liver transplantation as the only standard therapeutic option. Due to the increasing demand for transplantable liver, there is a persistent shortage of compatible liver donors. While tissue engineering approaches have emerged as a solution to regenerate damaged tissues, currently, the major limitations that hinder the clinical applications of bioengineered liver include hepatocyte maturation and vascularization. Developmentally, the liver is derived from the embryonic endoderm, which coemerges with the adjacent cardiac mesoderm to form the liver bud, indicating the importance of multilineage cellular interactions during organogenesis. In particular, cellular interactions between the hepatic and endothelial cells drive their differentiation and functional maturation. However, this process is not completely described due to the unavailability of complete in vitro model systems that recapitulate human liver development.

Materials and

Methods: : Towards this, we developed and characterized stem cell-derived hepatic and vascular organoids. We demonstrated the ability of these organoids to fuse and form vascularized hepatic assembloids. Using these assembloids, we aimed to recapitulate developmental interactions and cellular trajectories found during liver bud development. To elucidate how the stage-specific multilineage cellular interactions contribute to liver maturation and generation of unique populations, we performed single cell RNA sequencing (sc-RNA seq) of progenitor cells, unfused and fused hepatic and vascular assembloids over the course of differentiation. Functional assays were performed to validate the cell clusters and assess the secretion of cell-type specific proteins.

Results, Conclusions, and Discussions:: Preliminary data demonstrated that fusion enhances hepatocyte maturation and function, characterized by higher expression of hepatic markers, increased albumin (ALB) secretion, decreased alpha-fetoprotein (AFP) secretion, urea secretion, triglyceride storage, and enhanced cytochrome P450 3A4 (CYP3A4) activity, facilitating drug metabolism. Transcriptome data revealed that fusion led to the emergence of unique populations that expressed liver sinusoidal endothelial cell (LSECs) transcriptional signatures, suggesting tissue-specific endothelial specialization. Given the role of LSECs in the blood clotting cascade, these fused assembloids consisting of LSECs showed increased factor VIII (FVIII) activity, and FVIII secretion. Using labelled fibrinogen, we demonstrated that the conditioned media of fused assembloids was able to restore the fibrin clot formation of FVIII deficient plasma at levels comparable to the rhFVIII levels demonstrating the functional activity of secreted FVIII. Further, CellChat analysis leveraged to understand the signaling mechanisms activated in assembloid fusion showed that cluster 12 receives signals from VEGF, VISPLATIN, ANGPT and PLAU signaling pathways, suggesting that these pathways play a role in generation as well as maintenance of LSECs in the assembloids. Overall, a better understanding of how self-organizing vascularized liver tissue develops, will provide a strong molecular understanding of tissue maturation that can be used for developing more complex functional organoid models, paving the way for further advancements in developmental biology, bioengineered transplants, drug screening and disease modeling.

Acknowledgements and/or References (Optional)::