Single-cell morphological tracking of cell states to identify small-molecule modulators of liver differentiation.

We have developed a single-cell assay that combines Cell Painting-a morphological profiling assay-with trajectory inference analysis. We have applied this morphological trajectory inference to the bi-potent HepaRG liver progenitor cell line allowing us to track liver cell fate and map small-molecule-induced changes using a morphological atlas of liver cell differentiation. Our overarching goal is to demonstrate the potential of Cell Painting to study biological processes as continuous trajectories at the single-cell level, enhancing resolution and biological understanding. This work has identified small-molecule Src family kinase inhibitors that promote the differentiation of HepaRG cells toward a hepatocyte-like lineage as well as primary human hepatic progenitor cells toward a hepatocyte-like phenotype . These findings could significantly advance research on liver cell regeneration mechanisms and facilitate the development of cell-based and small-molecule therapies.