Abstract
Hepatocytes are crucial for drug screening, disease modeling, and clinical transplantation, yet generating functional hepatocytes in vitro is challenging due to the difficulty of establishing their authentic gene regulatory networks (GRNs). We have previously developed a two-step lineage reprogramming strategy to generate functionally competent human induced hepatocytes (hiHeps), providing an effective model for studying the establishment of hepatocyte-specific GRNs. In this study, we utilized high-throughput single-cell RNA sequencing (scRNA-seq) to explore the cell-fate transition and the establishment of hepatocyte-specific GRNs involved in the two-step reprogramming process. Our findings revealed that the late stage of the reprogramming process mimics the natural trajectory of liver development, exhibiting similar transcriptional waves of developmental genes. CD24 and DLK1 were identified as surface markers enriching two distinct hepatic progenitor populations respectively. Lipid metabolism emerged as a key enhancer of hiHeps maturation. Furthermore, transcription factors HNF4A and HHEX were identified as pivotal gatekeepers directing cell fate decisions between hepatocytes and intestinal cells. Collectively, this study provides valuable insights into the establishment of hepatocyte-specific GRNs during hiHeps induction at single-cell resolution, facilitating more efficient production of functional hepatocytes for therapeutic applications.