Modeling liver regeneration using 3D human liver organoids to assess the impact of ischemia on hepatic regeneration during transplantation

Abstract

Liver transplantation is the treatment of choice for end-stage liver disease. However, its success is often compromised by ischemia-reperfusion injury (IRI), a major contributor to graft dysfunction. Liver regeneration is a crucial process that enables recovery following injury and transplantation, involving progenitor cell activation, tissue remodeling, and terminal differentiation. Understanding how ischemia affects this regenerative response is essential for developing strategies that improve post-transplant outcomes.In this study, we established a liver regeneration model using undifferentiated 3D human liver organoids (hLiOs). To model ischemic injury, undifferentiated hLiOs were subjected to cold storage at 4°C for 16 hours. Organoids subjected to ischemic preconditioning, as well as non-ischemic controls, were systematically analyzed at pre- and post-differentiation stages through high-resolution microscopy, gene expression analysis, and immunofluorescence.Undifferentiated hLiOs expressed higher levels of LGR5 and EpCAM but lower levels of albumin, HNF4A, and CYP3A4 compared to primary hepatocytes. Conversely, upon differentiation, hLiOs showed decreased LGR5 expression, increased albumin secretion, and high expression of CYP3A4 and HNF4A. We then evaluated the impact of cold ischemia on the differentiation capacity of hLiOs. Differentiated hLiOs showed increased expression of hepatic functional markers, such as HNF4A, CYP3A4, and albumin, with significantly higher levels observed in organoids that had been exposed to ischemia prior to differentiation. In contrast, alpha-fetoprotein, a marker of hepatic progenitor activation, was more highly expressed in non-ischemic organoids. This pattern suggests that ischemic preconditioning may have accelerated or enhanced terminal hepatic differentiation.While IRI can impair regeneration and lead to liver failure, regeneration also acts as a defense mechanism against ischemic damage. Understanding this interplay is critical for improving outcomes after liver transplantation. Our preliminary findings indicate that ischemic injury can modulate the regenerative trajectory of liver organoids, promoting a shift from progenitor activation toward functional maturation. These results support the utility of hLiOs as a relevant in vitro model to study the complex interplay between ischemia and liver regeneration. This platform may aid in identifying therapeutic strategies to manage IRI and enhance hepatic recovery following transplantation.This research was funded by the Italian Ministry of Health, Ricerca Corrente.