Single-cell spatial transcriptomics reveals hepatocyte reprogramming in Fontan-associated liver disease

Fontan-associated liver disease (FALD) is a frequent complication in single-ventricle patients palliated with the Fontan operation. FALD severity can impact clinical decisions; however, the pathophysiology of FALD progression is unknown. Single-cell spatial transcriptomics (ST) was performed on liver explant tissue sections from FALD patients with early (n = 1) and advanced fibrosis (n = 1) using CosMx Spatial Molecular Imaging with in situ hybridization of 6000 genes. Immunofluorescence for liver zonation and cellular stress markers was performed to confirm protein expression based on ST analysis in additional FALD tissues (n = 18). Unbiased clustering yielded 12 liver cell types, comprising 6 subtypes of hepatocytes. FALD with advanced fibrosis demonstrated expansion of mid-zonal hepatocytes, accompanied by loss of zonal markers characteristic of canonical pericentral and periportal hepatocytes. A subset of hepatocytes in advanced FALD demonstrated increased cellular stress and a redundant zonal phenotype, which we have termed zonally ambiguous and stressed hepatocytes. CellChat analysis revealed that ectopic WNT2 signaling is likely driving disrupted hepatocyte zonation. To corroborate these bioinformatic findings, we performed immunofluorescent staining of FALD specimens, which confirmed a disruption of liver zonation, and a significant increase in heat shock protein 70 (HSP70). Lastly, HSP70 expression strongly correlated with the congestive hepatic fibrosis (CHF) score. Thus, single-cell ST has identified a population of hepatocytes with features of cellular stress and redundant zonal gene expression specific to advanced FALD. Further studies on hepatocyte metabolic function in Fontan patients will lead to a greater understanding of FALD development and progression during chronic maladaptation.