Fibrotic Phenotype in CRISPR knockout p53 of Hepatic Organoids within a Pro-Carcinogenic Microenvironment

Abstract

Abstract Hepatic fibrogenesis is a pathological outcome of chronic liver injury, characterized by the excessive accumulation of extracellular matrix proteins, leading to hepatocarcinogenesis. However, the lack of reliable models that mimic precancerous fibrogenesis in the early stage of the disease remains a significant obstacle. Here, we utilized human pluripotent stem cell-derived hepatic organoids (eHEPO) to replicate the early stages of human liver fibrosis, focusing on CRISPR/Cas9 system-mediated TP53 loss within a pro-carcinogenic microenvironment (pc-ME) comprising the secretome of activated hepatic stellate (LX2) and M2-polarized macrophages. We confirmed that our model represents an enrichment score across various signaling pathways according to transcriptome analysis, including inflammation, extracellular matrix (ECM) modification, fibrosis, and tumorigenesis. The model also displayed altered proliferation and differentiation properties depending on medium-derived stimulations, alongside noticeable alterations in key regulators of HIF1A, IFNA, STAT3, and Wnt/TGF-b signaling pathways. Importantly, our TP53KO-eHEPO model exhibited an enhanced fibrotic morphology with atypical cells, pseudo-glandular-tubular rosettes, steatohepatitis-like inflammatory areas, and ballooning-like hepatocytes. Additionally, we confirmed the augmentation of myofibroblast and fibrosis marker expression, including PDGFRB, COL1A1, COL3A1, and COL11A1, as well as early pro-carcinogenic markers GPC3 and MUC1. Overall, this model stands as a significant advancement in the study of liver fibrosis and hepatocarcinogenesis, offering a valuable tool for investigating the impact of first-hit genes like TP53 and inflammatory conditions on hepatic progenitor cell transformation in diverse microenvironments, and providing a potential platform for early-stage drug development and candidate identification.