Hepatocyte dedifferentiation in 2D culture reveals extensive transcriptomic and proteomic rewiring

Background: – Primary hepatocytes are commonly used in vitro to model liver metabolism, but prolonged culturing results in dedifferentiation and potentially limits the applicability of this model. Methods: – We characterized the transcriptome and proteome of full liver and primary hepatocytes as either freshly isolated cells or after 24 hours of 2D-culturing. Results: – We found that 2D-culturing for 24 hours changes more than 10, 000 genes and 3000 proteins compared with freshly isolated cells, accompanied by a decrease in transcriptional heterogeneity and a loss of zonal markers. Moreover, there were changes in proteins associated with the extracellular matrix, in mitochondrial and ribosomal protein abundances, as well as an increase in the abundance of acute-phase response proteins. Conclusion: – Collectively, primary mouse hepatocytes in culture rewire the transcriptome and proteome, which may affect the utility of this model to study physiological and molecular mechanisms related to the liver. We developed the Shiny app “Hepamorphosis” (https://cbmr.ku.dk/research/resources/shiny-apps/), which allows users to explore RNA/protein correlations, zonation profiles, and cell-type-specific transcription in full liver and cultured hepatocytes.