Leyao Zeng
Research Mentor(s): Robert Duncan
Mentor Department: Kresge Hearing Research Institute
Authors: Liqian Liu, R. Keith Duncan, Leyao Zeng
Session: Session 6 (3:00pm – 3:50pm)
Presentation Type: Poster 3
Abstract
Hearing and balance deficits are prevalent neurosensory disorders impacting quality of life. The damage and loss of function of hair cells in the cochlea and balance organs are the primary cause for these disorders. Unfortunately, these cells are known to have a limited ability to regenerate, and that rapidly declines after birth. New methods are required to derive inner ear-otic-cell types in highly controlled in vitro environments for downstream investigations into normal development, etiology of disease, and in regenerative medicine. Recent advances in the generation of stem cell-derived inner ear organoids open a new path for modeling inner ear biology, but these organoids are plagued by incomplete differentiation into otic phenotypes and extremely high heterogeneity. In this study, we seek to first generate high-purity otic progenitors and then introduce these into 3D organoid-like culture paradigms as a more reliable and reproducible method to generate inner ear-like tissues in vitro. High-purity otic progenitors were first generated and introduced into 3D organoid-like culture paradigms. 2D cultures of human embryonic stem cells (ESCs) were exposed to various concentrations of bone morphogenic protein (BMP4) or BMP-signaling inhibitor (LDN193189) to determine the conditions that maximize otic gene yield while minimizing other non-ectodermal fates. Primers were designed for each target gene using Primer3 and PrimerBlast. Primer efficiency in qPCR reactions was evaluated. The marker gene expression of BMP/LDN treated samples was then evaluated to determine the optimal condition for otic progenitor yield and validate otic marker expression with immunofluorescence to determine penetrance throughout the culture. Finally, these otic progenitors were introduced into 3D suspension culture under conditions that have been established to differentiate progenitors into inner ear sensory cells.
