Background: Liver transplantation (LTx) is the only curative treatment for end-stage liver disease. However, due to the shortage of high-quality deceased donor (DD) organs, 2,000 patients die annually while waiting for suitable livers. DD marginal livers (i.e. prolonged ischemic damage or fatty livers) could address this disparity, though it is associated with primary graft non-function and decreased recipient survival post-LTx. Advances in Normothermic Machine Perfusion (NMP) technology allow for extended preservation and ex-situ assessment of DD livers before transplant to guide decisions on liver quality and transplantability. Understanding the molecular mechanisms of injury, repair, and recovery during NMP is crucial for identifying safe transplant organs and potential pharmaceutical intervention targets to optimize marginal organ quality before transplant. This study aims to evaluate these mechanisms by analyzing temporal gene expression signatures using whole genome RNA sequencing in transplantable and non-transplantable livers during NMP.

Methods: Human donor livers without significant steatosis (n=6) and fatty donor livers (n=5) were subjected to 12 hours of NMP. Livers were categorized based on predefined criteria for demonstrating adequate hepatocellular function (transplantable vs. non-transplantable). Core needle biopsies were taken immediately before NMP and after 3 and 6 hours of NMP for bulk RNA sequencing. Bioinformatic analysis evaluated temporal changes in gene expression between functional and nonfunctional lean and fatty livers during NMP.

Results: Principal component analysis (PCA) revealed that gene expression changed for both fatty and lean livers during NMP. PCA effectively differentiated functional lean and fatty livers from non-functional fatty livers when all four categories were examined together. However, PCA was less sensitive in distinguishing functional lean and fatty livers from non-functional lean livers. The transcriptomic profile of biopsies from functional lean and fatty livers showed strong activation of mTORC1 signaling (growth/regeneration pathway) and protein secretion throughout NMP, correlating with improved hepatocellular function. Conversely, nonfunctional fatty livers exhibited a gradual decrease in the expression of both pathways during NMP.

Conclusion: We demonstrated that temporal overexpression of genes associated with liver regeneration and normal synthetic function during NMP can differentiate between transplantable and non-transplantable lean and fatty livers. Targeting the mTORC1 and protein secretion pathways may represent viable therapeutic interventions for rehabilitating the function of marginal livers, thus improving their quality and making them safe for transplantation. This can potentially reduce organ shortages. Our findings provide a foundation for future research aimed at predicting the recovery of marginal livers and enhancing liver transplantation outcomes through targeted therapies.