Long term expansion of Lgr5 positive supporting cells and differentiation into a hair cell-like phenotype from adult mouse derived cochlear organoids.

Introduction: Over 400 million people worldwide suffer from hearing loss, which requires medical intervention. The current treatments available to these patients are the use of hearing aids or cochlear implants. Although they improve the patients quality of life, 'normal' hearing is not completely restored. A primary cause of hearing loss results from damage to hair cells (HCs) due to excessive noise exposure, ageing, or the exposure to ototoxic drugs. These damaged HCs are not regenerated in humans and can continue to deteriorate with time. In non-mammalian vertebrates, supporting cells (SCs) in the cochlea have regenerative capacities and give rise to new HCs after damage, even in adulthood. Research with neonatal mice has also shown that SCs can give rise to new HCs after damage in vivo. Furthermore, neonatal SCs can be expanded and differentiated into hair cells in vitro. However, for translational purposes, it is important to evaluate if this is also possible with adult-derived tissue. Material and methods: Young adult and mature adult Lgr5GFP transgenic and C57CB/Bl6 mice were used for isolating cochleas. Cochleas were harvested and, after digestion with thermolysin and accumax, single cells were mixed with Matrigel, plated in 3D and grown in a high growth factor medium containing Wnt, R-spondin, and Noggin conditioned mediums. After passaging with trypsin and mechanical trituration, we evaluated proliferation and differentiation by immunofluorescence microscopy and qPCR or bulk sequencing. Results: Lgr5-expressing SCs derived from adult (p30-p200) Lgr5GFP transgenic and C57CB/Bl6 mice cochleas can be expanded to at least 9 passages without losing differentiation capacity. Lgr5 is highly expressed in expansion medium and lost after differentiation. Atohl expression increases during expansion and it is lost after differentiation. Myosin7 is not expressed in expansion medium and it is highly enhanced after differentiation. In parallel, we aim to setup a co-culture system where we will culture differentiated cochlear orga-noids with spiral ganglion neurons (SGN), to further mimic the in vivo physiology. This research will be the first step to building a functional primary cochlear organoid model to get a better understanding of the regenerative capacities of adult derived tissue. [ABSTRACT FROM AUTHOR]