A 3D Cell Culture Model Identifies Wnt/β‐Catenin Mediated Inhibition of p53 as a Critical Step during Human Hepatocyte Regeneration

The liver is a highly regenerative organ. While mature hepatocytes under homeostatic conditions are largely quiescent, upon injury, they rapidly enter the cell cycle to recover the damaged tissue. In rodents, a variety of injury models have provided important insights into the molecular underpinnings that govern the proliferative activation of quiescent hepatocytes. However, little is known about the molecular mechanisms of human hepatocyte regeneration and experimental methods to expand primary human hepatocytes (PHH). Here, a 3D spheroid model of PHH is established to study hepatocyte regeneration and integrative time‐lapse multi‐omics analyses show that upon isolation from the native liver PHH acquire a regenerative phenotype, as seen in vivo upon partial hepatectomy. However, proliferation is limited. By analyzing global promoter motif activities, it is predicted that activation of Wnt/β‐catenin and inhibition of p53 signaling are critical factors required for human hepatocyte proliferation. Functional validations reveal that activation of Wnt signaling through external cues alone is sufficient to inhibit p53 and its proliferative senescence‐inducing target PAI1 (SERPINE1) and drive proliferation of >50% of all PHH. A scalable 3D culture model is established to study the molecular and cellular biology of human hepatocyte regeneration. By using this model, an essential role of Wnt/β‐catenin signaling during human hepatocyte regeneration is identified.