Stable and functional human intestinal epithelium derived from induced pluripotent stem cells

Caco-2 cells are widely used in drug discovery but have limited drug metabolism, causing discrepancies with human physiology. This study evaluates human intestinal epithelial cells derived from pluripotent stem cell organoids, assessing morphology, gene expression, barrier function, transporter activity, drug metabolism, and cytotoxicity. These cells can be efficiently expanded in 2D culture, ensuring a stable supply of homogeneous, high-performance cells. Importantly, they retain small intestine-specific functions, such as high CYP3A4 activity and efficient peptide transport, over multiple passages. Their barrier integrity, transporter activity, and drug metabolism remain stable, making them a promising alternative to Caco-2 cells. Our findings support their use in microphysiological systems and organ-on-chip technologies, providing a physiologically relevant model for drug absorption, metabolism, and toxicity studies. These findings will aid in developing advanced in vitro models for the small intestine, improving drug absorption, metabolism, and toxicity studies. SIGNIFICANCE STATEMENT: This study shows that human pluripotent stem cell-derived intestinal epithelial cells retain key small intestine functions-drug metabolism, barrier integrity, and transporter activity-even after repeated division, offering a scalable and physiologically relevant alternative to Caco-2 cells.