Abstract
Degeneration or loss of inner ear hair cells (HCs) is irreversible and results in sensorineural hearing loss (SHL). Human-induced pluripotent stem cells (hiPSCs) have been employed in disease modelling and cell therapy. Here, we propose a transcription factor (TF)-driven approach using ATOH1 and regulatory factor of x-box (RFX) genes to generate HC-like cells from hiPSCs. Our results suggest that ATOH1/RFX1/RFX3 could significantly increase the differentiation capacity of iPSCs into MYO7AmCherry-positive cells, upregulate the mRNA expression levels of HC-related genes and promote the differentiation of HCs with more mature stereociliary bundles. To model the molecular and stereociliary structural changes involved in HC dysfunction in SHL, we further used ATOH1/RFX1/RFX3 to differentiate HC-like cells from the iPSCs from patients with myoclonus epilepsy associated with ragged-red fibres (MERRF) syndrome, which is caused by A8344G mutation of mitochondrial DNA (mtDNA), and characterised by myoclonus epilepsy, ataxia and SHL. Compared with isogenic iPSCs, MERRF-iPSCs possessed ~42–44% mtDNA with A8344G mutation and exhibited significantly elevated reactive oxygen species (ROS) production and CAT gene expression. Furthermore, MERRF-iPSC-differentiated HC-like cells exhibited significantly elevated ROS levels and MnSOD and CAT gene expression. These MERRF-HCs that had more single cilia with a shorter length could be observed only by using a non-TF method, but those with fewer stereociliary bundle-like protrusions than isogenic iPSCs-differentiated-HC-like cells could be further observed using ATOH1/RFX1/RFX3 TFs. We further analysed and compared the whole transcriptome of M1ctrl-HCs and M1-HCs after treatment with ATOH1 or ATOH1/RFX1/RFX3. We revealed that the HC-related gene transcripts in M1ctrl-iPSCs had a significantly higher tendency to be activated by ATOH1/RFX1/RFX3 than M1-iPSCs. The ATOH1/RFX1/RFX3 TF-driven approach for the differentiation of HC-like cells from iPSCs is an efficient and promising strategy for the disease modelling of SHL and can be employed in future therapeutic strategies to treat SHL patients.
Highlights
Degeneration or loss of inner ear hair cells (HCs) is irreversible and results in sensorineural hearing loss (SHL)
Our findings demonstrated that ATOH1/RFX1/RFX3 transcription factor (TF) could promote the differentiation of induced pluripotent stem cells (iPSCs)-derived HCs and facilitate the disease modelling of SHL using iPSCs from myoclonus epilepsy associated with ragged-red fibres (MERRF) patients with A8344G mutation of mitochondrial DNA
It has been suggested that the expression of Atoh[1] messenger RNA (mRNA) can be detected in otic progenitors (OPs) and the early immature HC stage differentiated from hESCs5, but not in HCs differentiated from mouse ESCs3
Summary
Degeneration or loss of inner ear hair cells (HCs) is irreversible and results in sensorineural hearing loss (SHL). In the regeneration of inner ear HCs in vitro, mouse bone marrow mesenchymal stem cells (MSCs). Official journal of the Cell Death Differentiation Association. Chen et al Cell Death and Disease (2018)9:437 were the first cell type to be differentiated into HC-like cells[1]. Mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been demonstrated to be differentiated into HC-like cells[2,3]. It has been suggested that using chicken utricle stromal cells as feeder cells for HC differentiation may make a subsequent examination problematic[4]. Ronaghi et al.[5] reported a feeder cell-free method for the generation of human ESC-derived HC-like cells, which exhibited many features of nascent HCs. Proneural Atoh[1] is a basic helix–loop–helix transcription factor (TF) and regulates the differentiation of HCs6
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