Transcriptomic Profile of Directed Differentiation of iPSCs into Hepatocyte-like Cells

The liver is the central organ in metabolism; however, modeling hepatic diseases remains limited by current experimental models. Animal models frequently fail to predict human liver physiology, while primary hepatocytes rapidly dedifferentiate in culture. We performed comprehensive transcriptomic profiling of induced pluripotent stem cells (iPSCs) differentiation into hepatocyte-like cells (HLCs) under two-dimensional (2D) and three-dimensional (3D) culture conditions. RNA sequencing analysis revealed the sequential activation of lineage-specific markers across major developmental stages: definitive endoderm (<i>FOXA2</i>, <i>SOX17</i>, <i>CXCR4</i>, <i>CER1</i>, <i>GATA4</i>), posterior foregut (<i>PROX1</i>, <i>GATA6</i>), and hepatoblasts (<i>HNF4A</i>, <i>AFP</i>). Comparative analysis demonstrated a markedly enhanced hepatic gene expression of 3D organoids, as demonstrated by a 33-fold increase in <i>HNF4A</i> expression and elevated levels of mature hepatocyte markers, including <i>ALB</i>, <i>SERPINA1</i>, and <i>UGT2B15</i>. However, the 3D cultures retained fetal characteristics (290-fold higher <i>AFP</i> expression) and exhibited significantly impaired metabolic function, with <i>CYP3A4</i> expression levels reduced by 2000-fold compared to the adult human liver. This partial maturation was further supported by a moderate correlation with adult liver tissue (ρ = 0.57). We demonstrated high reproducibility across five biologically distinct iPSCs lines, including those derived from patients with rare monogenic disorders. The establishment of quantitative benchmarks provides a crucial tool for standardizing in vitro liver models. Furthermore, we delineate the specific limitations of the current model, highlighting the need for further protocol optimization to enhance metabolic maturation and P450 enzyme activity. Functional validation of metabolic activity (CYP enzyme assays, albumin secretion) was not performed; therefore, conclusions regarding hepatocyte functionality are based on transcriptomic evidence.