iPSc-to-thymic epithelial progenitor differentiation under multifactorial DoE optimization and transcriptomic profiling

This study investigates the differentiation of human induced pluripotent stem cells (iPSc) into thymic epithelial progenitors (TEPs) through a combinatorial experimental design (Design of Experiments, DoE). Bulk RNA-seq was performed across 96 differentiation conditions covering key developmental transitions: definitive endoderm (DE), anterior foregut endoderm (AFE), third pharyngeal pouch endoderm (3PPE), and thymic epithelial progenitors (TEP). The multifactorial screen leveraged Plackett-Burman designs and transcriptomic similarity to in vivo pharyngeal development atlases to identify optimal differentiation cues. Overall design: RNA was extracted at the AFE, 3PPE and TEP stages of directed differentiation from iPSc across 96 conditions generated from three Plackett-Burman designs (36+36+24 runs), corresponding respectively to the DE_to_AFE, the AFE_to_3PPE and the 3PPE_to_TEP transitions. Each condition combined low/high concentrations of pathway modulators (BMP, WNT, FGF, TGFß, SHH, etc.) administered at defined time windows. Samples were sequenced using 3' RNA-seq (DGE-seq) and analyzed via bulk transcriptomics to evaluate proximity to gene signatures from developmental single-cell atlases.