Denis Kadilli
Research Mentor(s): Robert Duncan
Mentor Department: Kresge Hearing Research Institute
Authors: Denis Kadilli, LiQian Liu, Robert Duncan
Session: Session 6 (3:00pm – 3:50pm)
Presentation Type: Poster 2
Abstract
Damage to sensory hair cells in the inner ear leads to permanent hearing loss and balance disorders, often leading to sequelae such as social isolation, cognitive decline, and risk of falls. As such there has been considerable interest in developing regenerative medicine strategies to treat inner ear deficits. One recent advance is the generation of inner ear organs-in-a-dish, or so called organoids. Inner ear organoids are complex three-dimensional cultures that recapitulate the major developmental milestones from germ layer formation to otic intermediates to organoid cysts with sensory and nonsensory inner ear cell types. However, the organoid culture paradigms are exceptionally heterogeneous and must be optimized at every stage, particularly when formulations for commercial reagents change. In our study, we used mouse embryonic stem cell lines to examine the effects of an early cell-patterning morphogen–bone morphogenic proteins (BMPs)–on generation of non-neural ectoderm, otic placodal intermediates, and sensory hair cells. Specifically, we exposed embryoid bodies made from several different stem cell lines to different concentrations of BMP4 and examined the production of major biomarkers in the resulting tissue samples. To measure the variation and efficacy of different concentrations of BMP4, we cryosectioned frozen cell culture samples at various stages and stained with antibodies to ECAD and AP2 for nonneural ectoderm at 6 days in vitro (DIV6), ECAD, PAX2, and SIX1 for otic vesicle intermediates at DIV13, and SOX2 and MYOVIIA for sensory tissue in organoid cysts at DIV20 and after. We found significant variation in patterning depending on BMP4 concentration and that this was cell line dependent, suggesting that endogenous BMP signaling varies substantially between the cell lines. Ultimately, we identified optimal BMP4 concentrations to reliably drive the production of hair cells and established a pipeline for validating this critical step in the inner ear organoid protocol.
