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

Abstract

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.

Differentiation of embryonic stem cells into a putative hair cell-progenitor cells via co-culture with HEI-OC1 cells

“…2 B). On the other hand, EBs after 14 days of contact co-culture exhibited changes in morphology, showing several protruding structures from the surface, which resembled generated otic organoids from a prior report 13 . Each cellular structure generated from contact co-culture of dissociated mESCs or EBs expressed GFP, and Myo7a and Sox2 markers (Fig.…”

“…There are several things to be cleared before the actual usage of stem cells for transplantation. First, we have to differentiate this cell further to have complex functions such as the mechanoelectric transduction of hair cell-like cells, as confirmed by previously reported paper showing electric signal induced by the developed cells 14 and expression of specific marker FM1-43 in our recent publication 13 . However, it is yet to replicate the fully functional specific cells that are capable of sound transduction.…”

“…The most straightforward and common way to differentiate ESCs is into hair cells is via well-timed and gradual addition of bulk molecules. Conventional differentiation protocols for the generation of inner ear hair cells from stem cells in vitro are multi-step and require the well-timed addition of several exogenous bioactive molecules, such as BMP4, transforming growth factor β inhibitor, FGF2, and Wnt 13 – 15 . We posited that co-culture using HEI-OC1 cells could induce otic differentiation of EBs.…”

Effects of prenatal photobiomodulation treatment on neonatal hypoxic ischemia in rat offspring

“…Lasers with a wavelength of 808 nm have demonstrated deepest tissue penetration and, as a result, have been widely used for various brain disorders 1 , 2 , 26 , 47 , 74 , 75 . Numerous cells/tissues responded to PBM, including neurons 76 , microglia 77 , astrocytes 78 , embryonic rat brains 79 , mouse embryonic stem cells 80 , and chicken embryos 81 . Furthermore, 808 nm low-level laser light penetrated the rabbit skull and brain to a depth of 2.5-3 cm when placed upon the skin surface in previous reports 82 - 84 .…”

Application of Mesenchymal Stem Cell Therapy and Inner Ear Regeneration for Hearing Loss: A Review

“…Using light-emitting diodes (LEDs) with a wavelength of 630 nm, the differentiation of hair cell-like cells derived from mouse embryonic stem cells (ESCs) was coupled with the overexpression of reactive oxygen species (ROS). A transcriptome analysis revealed the factors correlated with the effect of PBM on the formation of otic organoids [21].…”