Epithelial Cell Differentiation from Human Induced Pluripotent Stem Cells Using a Single-Cell Culture Method

Abstract

: The conventional culture method of human induced pluripotent stem cells (hiPSCs) has been performed in colony cultures using feeder cells, such as mouse embryonic fibroblasts, which require setup times and procedural complexity, a potential risk of transmission of animal pathogens. Besides, the colony culture exhibits slow growth rate and often give rise to heterogeneous cellular states. However, developing technical methodologies of hiPSCs remains pivotal for use in medical applications and research. Here, we investigated whether hiPSCs passaged and expanded as single cells under feeder-free conditions could differentiate into ectodermal-epithelial cells as a source of cells for future regenerative medicine research. First, an hiPSC line 253G1 was cultured in colonies maintained on feeders and subsequently transferred to a single cell on feeder-free. hiPSCs were then cultured as single cells for 28 days in an induction medium supplemented with retinoic acid, bone morphogenetic protein 4, and N2 supplement for epithelial cell differentiation. The expression of epithelial markers, tumor protein p63 (P63), cytokeratin (CK) 18, and CK14 in induced cells was evaluated over time using real-time polymerase chain reaction, western blotting, and immunocytochemistry. Results showed that hiPSCs cultured as single cells ex pressed pluripotency markers, as evidenced by colony cultures maintained on feeders. On day 7 post-induction, hiPSCs as-sumed a cobblestone-like morphology in the epithelial induction medium. Induced cells displayed increased mRNA expression levels of CK18 , P63 , and CK14 during the 28-day induction period. Furthermore, the expression levels of CK18, P63, and CK14 were detected via western blotting and immunocytochemistry. Our findings suggest that hiPSCs cultured as single cells could be differentiated into epithelial cells.