Supplementary Material for: Media comparisons for the differentiation of human iPSCs into the otic lineage

Introduction Otic organoids differentiated from human pluripotent stem cells are three-dimensional in vitro cultures that broadly mimic the complexity and functionality of the human inner ear. They provide a valuable model for developmental and disease-related studies. However, current protocols differ substantially in efficiency and reproducibility. In this study, we investigated whether different stem cell maintenance media influence the differentiation of keratinocyte-derived induced pluripotent stem cells (kiPSCs) into the otic lineage. Methods Keratinocyte-derived iPSCs were cultured in either a self-made FTDA medium or the commercially available PeproGrow™ hESC medium and subsequently subjected to an established otic differentiation protocol. Early developmental stages, including the pre-placodal region, the otic placode, and pro-neural sensory regions, were analysed using immunofluorescence and gene expression profiling. Results While no significant differences were observed in iPSC maintenance or pluripotency between the two media, distinct differences emerged during otic differentiation. Media composition influenced the expression of placodal, otic, and pro-sensory markers at multiple stages, indicating differential responsiveness to otic induction cues. Conclusion Our findings demonstrate that stem cell maintenance media composition is a critical determinant of subsequent otic lineage differentiation. These results provide guidance for optimizing stem cell culture conditions and improving the reproducibility of otic organoid differentiation protocols.