Critical role of FGF, RA, and WNT signaling for otic lineage specification from human PSCs in a 2D/3D-combined differentiation system

The otic placode, from which the inner ear develops, initially forms as a thickened ectodermal patch adjacent to the dorsolateral hindbrain. Induction of otic placodal cells from human pluripotent stem cells (hPSCs) provides a robust approach to investigation of otic development. However, attempts to recapitulate otic lineage specification from hPSCs by stepwise differentiation methods have had limited success. One possible reason is that the role of signaling pathways in otic cell differentiation is not fully understood. Here, we developed a novel differentiation system involving use of suspension culture (3D floating culture) in combination with signaling factors for generating otic placodal cells via stepwise differentiation of hPSCs. We demonstrate that hPSC-derived pre-placodal cells acquired the potential to differentiate into posterior placodal cells after fibroblast growth factor 2 (FGF2) and retinoic acid (RA) treatment. Subsequent activation of WNT signaling following posterior placode specification induced differentiation of SIX1+/PAX8+/SOX2+ otic placodal cells. qPCR analysis showed that WNT activation increased expression of otocyst markers, including PAX8, PAX2, DLX5 and FBXO2. Moreover, induced otic cells exhibited similar gene expression patterns to the ventral and medial portions of the otocyst that give rise to the sensory epithelium of the inner ear. Collectively, our results indicate a critical role for FGF2, RA, and WNT signaling for the in vitro differentiation of the otic cell lineage from hPSCs. Our new culture system paves the way for improved modeling of early human inner ear development and will be of value to studies on the further differentiation of sensory epithelial cells. Overall design: RNA seq of human placodal cells