Generation and characterization of an otic bioengineered neuronal organoid (oBENO) from human pluripotent stem cells

by Angeliki Koufali
Doctoral thesis
Date of Examination:2025-05-07
Date of issue:2025-12-03
Advisor:Dr. Maria-Patapia Zafeiriou
Referee:Dr. Maria-Patapia Zafeiriou
Referee:Prof. Dr. Argyris Papantonis
Persistent Address: http://dx.doi.org/10.53846/goediss-11680

 

 

Files in this item

Name:Dissertation_Angeliki Koufali.pdf
Size:10.5Mb
Format:PDF

This file will be freely accessible after 2026-05-06.

Abstract

English

Hearing loss impacts nearly 20% of the global population, and according to estimations its prevalence will increase by 2050, affecting around 1 in 4 individuals. The most common form of hearing impairment, sensorineural hearing loss (SNHL), is traced back to the loss of hair cells (HCs), the mechanosensory cells that detect sound and movement, which lack regenerative potential in vertebrates. HC loss can in turn lead to spiral ganglion neuron (SGN) death, which form transfer auditory input to the auditory cortex. Although animal models remain indispensable for elucidating hearing loss mechanisms, the evolutionary gap between rodents and humans can hinder the translatability of preclinical data. In this study, we generated a robust human induced pluripotent stem cell (hiPSC)-derived organoid model that reproducibly develops HCs and SGNs, the otic bioengineered neuronal organoid (oBENO). The generation of oBENO relies on the patterning of BENO with specific developmental cues towards an otic fate. Moreover, we employed genome engineering of hiPSCs to trace and characterize HCs and SGNs in the developing oBENO. Temporal bulk RNA sequencing analysis of oBENOs, between days 0 and 60, revealed that the oBENO development closely mimics the in vivo stages of early inner ear formation. Moreover, we discovered the development of several inner ear specific supporting cell populations, and an intricate vascular network, underscoring the cellular complexity of the oBENO model. We introduced two reporter lines for the genetic tracing of HCs (trHC) and SGNs (trSGN) that allowed the live monitoring of the labelled cells and analysis of their functional properties, complementing our patch clamp analysis of AAV-transduced oBENOs. Calcium imaging revealed that SGNs in oBENO can be depolarized by exogenous glutamate and nicotine, in line with in vivo data. Importantly, we identified that glutamate neurotransmission is responsible for SGN activation in oBENO. Furthermore, we explored the versatility of the oBENO system in approaches focused on improving hearing restoration therapies. We successfully enhanced hair cell differentiation in oBENOs generated by inducible lines and demonstrated that oBENO can serve as a valuable AAV screening platform. By laying the groundwork for a human inner ear in vitro model, the oBENO could offer a platform for unveiling the mechanisms of inner ear development and pathology. This hiPSC-derived organoid model could complement animal in vivo model studies and accelerate the progress in disease modelling, high-throughput drug screenings and regenerative medicine.
Keywords: inner ear; otic organoid; human; pluripotent stem cells; hair cell; spiral ganglion neuron; reporter line