Abstract: Novel inner ear organoid models for studying hair cells in normal development and in the deaf-
blindness disease Usher Syndrome Type 1F
The fundamental human sense of balance, which comprises both movement through the world and one’s body
position in space, begins with specialized biological detectors called “hair cells” responsible for converting
mechanical stimuli into electrical signals (also known as mechanotransduction). Hair cells perform this function
by converting mechanical stimuli on their apical surface (received by a specialized structure of actin-rich
stereocilia) into electrical impulses on their basolateral surface (produced by specialized ribbon synapses).
Despite their critical role and decades of study in animal models, many aspects of the hallmarks of hair cell
development specifically in developing human tissue have yet to be explored. To circumvent the difficulty in
access, collection, and preservation of in vivo or ex vivo human tissue, human induced pluripotent stem cell
(hiPSC) derived inner ear organoids (IEOs) have emerged as a new model for studying human development.
IEOs are 3D aggregates of cells containing many internal vesicles lined with inner ear sensory epithelia and
synapsing neurons. Strong histologic and limited transcriptomic data of IEOs show that the in vitro organoids
recapitulate many of the diverse features found in the in vivo inner ear. Unfortunately, the sensory cells of interest
are buried by a large population of supporting and mesenchymal cells, preventing their long-term developmental
and functional study. My recent work in the lab has shown that perturbation of extracellular matrix proteins
reverses the polarity of the sensory epithelia such that the progenitor epithelia remain on the surface and have
their apical surfaces pointed outwards into the media. The outwardly facing hair cells with this new method are
now both accessible and imageable, overcoming the limitations of previous IEO versions. Based on initial
characterization, I hypothesize that inverted apical-out IEOs yield more numerous and more functionally mature
hair cells. In Aim 1, I will characterize the genetic and physiologic properties of the human hair cell population
that is now accessible through our in vitro model. I will perform single cell RNA sequencing in the mature organoid
and bathe the organoids in artificial solutions that mimic in vivo endolymph to attempt to mature the transcriptomic
and electrophysiological profiles of the hair cells. In Aim 2, I will use a patient-derived hiPSC line from a patient
with a devastating genetic deaf-blindness mutation known as USH1F to investigate the development of critical
apical structures and the disrupted effects that damage hair cell function. I will then attempt to use viral-mediated
gene therapies before the onset of hair cell dysfunction to restore proper hair cell development. These
experiments will reveal the first generation of many applications of this new approach to generate apical-out
inner ear organoids in order to study human development, disease progression, and as a platform to develop
novel therapeutics.
