Ohman Kwon^1,†, Kwang Bo Jung^1,2,†, Kyeong-Ryoon Lee^3,†, Ye Seul Son^1,2, Hana Lee^1,2, Jong-Jin Kim^1,2, Kwangho Kim¹, Seop Lee^3,4, Yoo-Kyung Song³, Jaeeun Jung^1,2,Kunhyang Park¹, Dae-Soo Kim^1,2, Myung Jin Son^1,2, Mi-Ok Lee^1,2, Tae-Su Han¹, Hyun-Soo Cho^1,2, Soo Jin Oh⁵, Haeun Chung^6,7, Sang-Heon Kim^6,7, Kyung-Sook Chung^1,2, Janghwan Kim^1,2,*, Cho-Rok Jung^1,2,* and Mi-Young Son^1,2,*

¹Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea.
²KRIBB School of Bioscience, Korea University of Science and Technology, Daejeon 34113, Republic of Korea.
³Laboratory Animal Resource Center, KRIBB, Ochang, Chungbuk 28116, Republic of Korea.
⁴College of Pharmacy, Chungnam National University, Daejeon 34134, Republic of Korea.
⁵Asan Institute for Life Sciences, Asan Medical Center and Department of Convergence Medicine, College of Medicine, University of Ulsan, Seoul 05505, Republic of Korea.
⁶Center for Biomaterials, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea.
⁷Division of Bio-Medical Science and Technology, KIST School, Korea University of Science and Technology, Seoul 02792, Republic of Korea.

^*Corresponding author.
^†These authors contributed equally to this work.

Advanced technologies are required for generating human intestinal epithelial cells (hIECs) harboring cellular diversity and functionalities to predict oral drug absorption in humans and study normal intestinal epithelial physiology. We developed a reproducible two-step protocol to induce human pluripotent stem cells to differentiate into highly expandable hIEC progenitors and a functional hIEC monolayer exhibiting intestinal molecular features, cell type diversity, and high activities of intestinal transporters and metabolic enzymes such as cytochrome P450 3A4 (CYP3A4). Functional hIECs are more suitable for predicting compounds metabolized by CYP3A4 and absorbed in the intestine than Caco-2 cells. This system is a step toward the transition from three-dimensional (3D) intestinal organoids to 2D hIEC monolayers without compromising cellular diversity and function. A physiologically relevant hIEC model offers a novel platform for creating patient-specific assays and support translational applications, thereby bridging the gap between 3D and 2D culture models of the intestine.