Background : Hepatocellular carcinoma (HCC) poses challenges for direct examination in patient samples due to its molecular complexity, heterogeneity, and limited accessibility. Employing hepatic progenitor cells, we replicate genetic changes in HCC, providing insights into tumor initiation and progression. Previous studies have demonstrated that introducing TP53 and BAP1 mutations into human chemically derived hepatocytes (hCdHs) results in a cholangiocarcinoma cell line. While Sox9 is expressed in hepatocytes, the precise roles of Sox9+ hepatocytes in HCC remain elusive. Our research investigates the impact of SOX9 gene knockout in HCC context.

Methods : Human primary hepatocytes were reprogrammed using a medium supplemented with HGF, A83-01, and CHIR99021, yielding hCdHs. Sequential CRISPR/Cas9-mediated mutations were introduced into TP53, BAP1, and SOX9 genes. The resultant cell line was utilized to generate organoids. Subsequently, analysis of the cell line was conducted through real-time PCR.

Results : The new cell line exhibited downregulated SOX9 mRNA expression. Hepatocyte markers (HNF4α & AFP) were upregulated, while HCC marker ARID1A was downregulated, and CD44, HCC stem cell marker, was upregulated. Cholangiocyte markers (EpCAM & Ck19) and extrahepatic cholangiocarcinoma markers (SOX4, SOX17, & Jag1) were downregulated. Taken together, these results suggest a shift toward hepatocyte-like properties. New cell-derived organoids exhibited diverse morphologies, unlike the control organoids, while maintaining a PCR profile consistent with the 2D cell line, confirming the preservation of their cellular nature.

Conclusions : This preliminary data highlights the potential of CRISPR/Cas9 technology for generating an HCC disease model using hCdHs. Further investigations are essential to validate the true characteristics of this new cell line.