UC San Diego

The loss of hair cells (HCs) and spiral ganglion neurons (SGNs) in the inner ear can lead to irreversible hearing loss in mammals. Because these cells are few in number and difficult to access, studies on their development, degeneration, and reprogramming capabilities in response to injury have been limited. Previous studies have analyzed the gene expression profiles of HCs and SGNs in mice through bulk and single-cell RNA sequencing, with some identifying transcription factor (TF) genes, such as Atoh1, Pou4f3, Gfi1, Neurog1, and Neurod1 as significant for HC and SGN development and maturation. However, it is not known whether these findings apply to the human system. Thus, a reliable and accurate in vitro model of human HCs and SGNs has yet to be generated to study their development and injury responses. In this study, we overexpressed two unique sets of TF gene cassettes in IMR90.4 cells with an endogenous POU4F3-p2A-eGFP reporter: (1) TetO-ATOH1-p2A-POU4F3-p2A-GFI1 (APG1) and (2) TetO-NEUROG1-p2A-ATOH1-p2A-NEUROD1 (NAN1) for generation of HC- and SGN-like cells, respectively. These modified hiPSCs were differentiated and collected for bulk RNA-sequencing after 6 and 14 days. Sequencing analysis revealed that after six days, APG1 induced cell gene expression was enriched for auditory processes, but this enrichment was less significant by day 14. Though day 6 NAN1 induced cells appear more neuronal than day 6 APG1 cells, possibly leading to an SGN-like cell fate, they do not take on a definitive cell type identity, instead expressing some HC- and neuronal genes at days 6 and 14.