Single Cell Analysis Reveals the Presence of Novel Intermediate Cells in Both Mice and Patients With Severe MASLD

: The landscape of cellular abnormalities occurring during metabolic dysfunction-associated steatotic liver disease (MASLD) is not completely clarified. We investigated cell heterogeneity involved in progressive MASLD at single cell resolution in mice fed AMLN. Single cell RNA sequencing (Sc-RNAseq) and spatial proteomics were applied to decipher cell populations and genes/pathways guiding MASLD progression. We identified 32 clusters, including hepatocytes (HEPs), hepatic stellate cells (HSCs), endothelial cells (ENDOs), Kupffer cells (KCs), and immune cells. HEPs clusters changed across disease severity, acquiring a periportal localization in MASH-fibrosis. The latter conditions were featured by ENDO clusters with higher expression of extracellular matrix (ECM) molecules, resident and recruited KCs/immune clusters with M1 polarization and HSCs with a myofibroblast phenotype. Finally, we highlight 5 hybrid populations named HSCs/ENDOs, HEPs/ENDOs, KCs/ENDOs, and HEPs/KCs which were confirmed by spatial proteomics across disease stages in mice and MASLD patients. In sum, we observed an evolution of cellular heterogeneity during MASLD and identified novel intermediate populations that feature advanced disease.