Human Pluripotent stem cells-derived Inner ear organoids recapitulate otic development in vitro

The molecular characterization of early stages of human inner ear development is limited by the difficulty in accessing samples at early gestational stages. Some aspects of inner ear morphogenesis can be recapitulated using pluripotent stem cell directed differentiation in inner ear organoids (IEOs). Once validated and benchmarked, these models could provide a unique tool to complement and refine our understanding of human otic differentiation and could be used to model developmental defects.Here we provide a first characterization of early human embryonic otocyst development and compare the primary tissue to the iPSC-derived inner ear cell types. Multiplex immunostaining and single cell RNA sequencing were used to characterize human iPSC-derived IEOs at 3 key developmental steps, providing a new and unique signature of in vitro derived otic- placode, epithelium, neuroblasts and sensory epithelia. The expression and localization of key markers were further evaluated in human embryos. We show that the otic placode derived in vitro (day 8-12) matches marker expression of Carnegie Stage (CS) 11 embryos, and subsequently (day 20-40) gives rise to otic epithelia and neuroblasts comparable to the CS13 embryonic stage. Differentiation of sensory epithelia, including supporting cells and hair cells, starts in vitro at day 50-60 of culture. The maturity of these cells is equivalent to vestibular sensory epithelia at week 10 or cochlear tissue at week 12 of development, prior to functional onset. Taken together these data indicates that the current state of the art protocol enables the specification of bona fide otic tissue, supporting further application of IEOs to model inner ear biology and disease Overall design: We have analyzed by transcriptional profiling 3 steps of otic development in inner ear organoids obtained through directed differentiation of iPSC in 3D culture. 1: day 8 of culture: otic placode stage; 2: day 26 of culture: otic vesicle stage; 3: day 60 of culture: sensory epithelia development. Cells collected at day 8 were obtained from 200 unsorted organoids. This provides an overview of the efficiency of otic placode induction. In addition to otic placode, other tissues co-develop including: epidermis, mesenchyme, neural crest and neural epithelium. A small percentage of placode neurons is also detected. Cells collected at day 26 were obtained from 100 organoids. After trituration cells were SORTED using EPCAM/CD326 antibody staining. Only the EPCAM+ fraction was processed for scRNAseq. This enable to identify the otic epithelium (OP) as well as otic neuroblasts (ONB) derived from delamination from the former. In addition, EPCAM+ epidermis can also be identified. Small populations of mesenchyme and neural epithelia cells are purified and identified. Cells harvested at day 60 were obtained from 2 independent experiment. EXP1: 48 organoids (unselected) were collected and SORTED using EPCAM/CD326 antibody staining. EXP2: 8 organoids (selected based on visual assessment of GFP positive vesicles) were collected and SORTED using EPCAM/CD326 antibody staining. In both cases the EPCAM+ pool were sequenced. Beside otic epithelium (OEP) and Epidermis (EP), other co-developed tissues (mesenchyme and neural epilelia etc) were identified. The same 3 stages of otic development are characterized in parallel by immunostaining and compared to human tissue and the equivalent stage of differentiation. This study a fits comparison of inner ear organoid differentiation from human pluripotent stem cells and human primary tissue.