Abstract
Math1, a basic helix-loop-helix transcription factor homolog of the Drosophila atonal gene, is considered to be a key factor for induction of sensory hair cells (HCs) during development of the organ of Corti or cochlea. Although embryonic stem (ES) cells are able to produce HC-like cells, the role of Math1 in induction of those cells has not been thoroughly elucidated. In the present study, we introduced Math1 into ES cells in order to achieve efficient generation of HC-like cells. ES cells carrying Tet-inducible Math1, Math1-ES cells, were generated using a Tet-On gene expression system. Embryoid bodies (EBs) formed in the absence of doxycycline (Dox) for 4 days were allowed to grow for an additional 14 days in the dishes in the presence of 400 μg/ml of Dox. At the end of those 14-day cultures, approximately 10% of the cells in EB outgrowths expressed the HC-related markers myosin6, myosin7a, calretinin, α9AchR, and Brn3c (also known as Pou4f3) and showed formation of stereocilia-like structures, whereas few cells in EB outgrowths grown without Dox showed those markers. Reporter assays of Math1-ES cells using a Brn3c-promoter plasmid demonstrated positive regulation of Brn3c by Math1. Furthermore, such HC-related marker-positive cells derived from Math1-ES cells were found to be incorporated in the developing inner ear after transplantation into chick embryos. Math1-ES cells are considered to be an efficient source of ES-derived HC-like cells, and Math1 may be an important factor for induction of HC-like cells from differentiating ES cells.
Highlights
Molecular mechanisms related to hair cells (HCs) development have been elucidated in detail,[9,10,11,12] while the transcription factor Math[1] has been identified as the earliest HC-specific gene required for definitive HC development.[13,14] Loss of Math[1] results in complete failure of HC differentiation in the mouse cochlea,[15] while its overexpression via viral vectors was reported to generate new HCs16,17 and substantially improve hearing thresholds in adult deaf guinea pigs.[18]
We investigated the effects of Math[1] overexpression on HC differentiation of Embryonic stem (ES) cells
Reporter assays of Math1-ES cells using a Brn3c-promoter plasmid demonstrated positive regulation of Brn3c by Math[1]. We found that such HC-related marker-positive cells derived from Math1-ES cells were incorporated in the developing inner ear after transplantation into chick embryos
Summary
Molecular mechanisms related to HC development have been elucidated in detail,[9,10,11,12] while the transcription factor Math[1] ( known as Atoh1) has been identified as the earliest HC-specific gene required for definitive HC development.[13,14] Loss of Math[1] results in complete failure of HC differentiation in the mouse cochlea,[15] while its overexpression via viral vectors was reported to generate new HCs16,17 and substantially improve hearing thresholds in adult deaf guinea pigs.[18] it has been reported that introduction of Math[1] led to successful generation of HC-like cells from bone marrow mesenchymal stem cells.[19] Math[1] seems to be a key transcription factor for HC development. We found that such HC-related marker-positive cells derived from Math1-ES cells were incorporated in the developing inner ear after transplantation into chick embryos. Math1-ES cells are considered to be an efficient source of ES-derived HC-like cells, and Math[1] is an important factor for induction of HC-like cells from differentiating ES cells
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