Trajectory analysis of hepatic stellate cell differentiation reveals metabolic regulation of cell commitment and fibrosis

Abstract Defining the trajectory of cells during differentiation and disease offers the possibility to understand the mechanisms driving cell fate and identity. However, trajectories of human cells are largely unexplored. By investigating the proteome trajectory of iPSCs differentiation to hepatic stellate cells (dHSCs), we identified RORA as a key transcription factor governing the metabolic reprogramming of HSCs necessary for HSCs’ commitment, identity, and activation. Using RORA deficient iPSCs and pharmacologic interventions, we showed that RORA is required for mesoderm differentiation and prevents dHSCs activation by reducing the high energetic state of the cells. While RORA knockout mice had enhanced fibrosis, RORA agonists rescued multi- organ fibrosis in in vivo models. RORA expression was consistently found to be negatively correlated with liver fibrosis and HSCs activation markers in patients with liver disease. This study reveals that RORA regulates cell metabolic plasticity, crucial for mesoderm differentiation, pericyte quiescence, and fibrosis, influencing cell commitment and disease mechanisms. Summary This study describes the trajectory of induced pluripotent stem cells (iPSCs) differentiation to hepatic stellate cells (dHSCs). We identify RAR-related orphan receptor alpha (RORA) as a transcription factor essential for mesoderm commitment and dHSCs identity and fibrogenic activation by regulating metabolic plasticity.