Enhanced Inner-Ear Organoid Formation from Mouse Embryonic Stem Cells by Photobiomodulation

Photobiomodulation (PBM) stimulates different types of stem cells to migrate, proliferate, and differentiate in vitro and in vivo. However, little is known about the effects of PBM on the differentiation of embryonic stem cells (ESCs) toward the otic lineage. Only a few reports have documented the in vitro differentiation of ESCs into inner-ear hair cells (HCs) due to the complexity of HCs compared with other target cell types. In this study, we determined the optimal condition to differentiate the ESCs into the otic organoid using different culture techniques and PBM parameters. The efficiency of organoid formation within the embryoid body (EB) was dependent on the cell density of the hanging drop. PBM, using 630 nm wavelength light-emitting diodes (LEDs), further improved the differentiation of inner-ear hair cell-like cells coupled with reactive oxygen species (ROS) overexpression. Transcriptome analysis showed the factors that are responsible for the effect of PBM in the formation of otic organoids, notably, the downregulation of neural development-associated genes and the hairy and enhancer of split 5 (Hes5) gene, which inhibits the differentiation of prosensory cells to hair cells. These data enrich the current differentiation protocols for generating inner-ear hair cells.