The Molecular Landscape of Liver Transplant Ischemia-Reperfusion Injury: Insights into Tissue Repair, Regeneration and Outcome Risk Stratification - Liver transplantation (LT) is the standard treatment for end-stage liver disease, but organ shortages have increased reliance on expanded criteria donors (ECDs) such as DCD, aged, and steatotic livers, which are more prone to ischemia-reperfusion injury (IRI) and poorer outcomes. Many transplantable livers are discarded due to the lack of reliable tools to assess quality and predict function. While cold static preservation (CSP) limits evaluation, normothermic machine perfusion (NMP) allows dynamic assessment and may improve ECD use. However, it remains unclear which livers benefit most from NMP, or which IRI-protective mechanisms are targetable. To address these gaps, we will leverage two ongoing biobanks: Cohort 1 (UMB, n=242) includes CSP-preserved livers with biopsies at procurement (L0), pre-implantation (L1), and post-reperfusion (L2), with >5 years of follow-up; Cohort 2 (Toronto, n=85) includes DCD and ECD livers with L0, pre-/post-NMP, and L2 samples, with >2 years of follow-up. Overall Hypothesis Donor liver cell states, donor-recipient cellular interactions and linked cell-type specific molecular profiles serve as robust predictors of inflammatory responses and maladaptive repair circuits, thus influencing liver injury and graft outcomes. Aim 1. Assess the influence of donor quality on post-transplant liver graft function by comprehensively examining the composition and functional characteristics of liver donor cells preserved under two distinct conditions. Hypothesis: Cell types, cell type- specific transcriptomes, and gene regulatory networks (GRNs) in donor livers reflect quality and preservation status providing crucial insights into the initial mechanisms of injury and repair that influence post-LT function. Sub Aim 1.a: Combined single nucleus (sn)RNAseq/snATACseq will be used to identify the cell types and cell type-specific pathways that characterize ECD livers (CSP and NMP). Sub Aim 1.b: By integrating paired perfusate and liver single-cell data, we aim to identify non-invasive, cell-type-specific markers of graft injury and function. Aim 2. Identify cell type-specific pathways and regulatory gene networks associated with the severity of IRI and elucidate the mechanisms that determine graft outcomes in CSP & NMP preserved livers. Hypothesis: The transcriptional and epigenetic landscape of the donor liver determines the severity of IRI, influencing dynamic cell type-specific molecular pathways, affecting graft outcomes. Paired L1 (CSP)/post-NMP-L1 and L2 snRNAseq/snATACseq biopsies from ECD livers will be evaluated. Aim 3. Develop a comprehensive Liver Quality Risk Stratification Index (LQRSI) for predicting graft function. Hypothesis: Integrating donor molecular markers with donor and recipient clinical and demographic data will improve prediction of graft outcomes. Guided by snRNA-seq data (SA1.a), we will use bulk mRNA from pre-implantation biopsies to identify cell type–specific donor quality markers. Three models 1) donor clinical data, 2) molecular profiles, and 3) recipient characteristics, will be integrated into a composite LQRSI using late fusion, adjusted for preservation type, and evaluated via cross-validation, AUC, and concordance. qPCR will validate findings in independent and prospective cohorts.
