ATOH1/RFX1/RFX3 transcription factors facilitate the differentiation and characterisation of inner ear hair cell-like cells from patient-specific induced pluripotent stem cells harbouring A8344G mutation of mitochondrial DNA

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.

Elimination of Mutant mtDNA by an Optimized mpTALEN Restores Differentiation Capacities of Heteroplasmic MELAS-iPSCs

“…However, some reports have demonstrated that iPSCs from patients with mitochondrial diseases, who carry various types of mtDNA mutations, show differentiation defects in vitro . 14 , 15 , 28 In this study, MyoD-iPSC lines (#21-6 and #25-3) with high proportions (≥96.1%) of G13513A mutant mtDNA showed poor differentiation into αSMA-positive cells and TUBB3-positive neuronal cells using a method of spontaneous EB-mediated differentiation. A previous study has demonstrated that iPSCs derived from patients with MELAS, who carried ∼100% of A3243G mutant mtDNA, could spontaneously differentiate into αSMA-positive cells and Tuj1-positive neuronal cells.…”

Age-related transcriptome changes in Sox2+ supporting cells in the mouse cochlea

“…Rfx1 has an important function in brain tumors and sensorineural hearing loss. Together with Atho1 , Rfx1/3 can induce HC-like cell differentiation, and the conditional knockout of Rfx1/3 leads to severe hearing loss and OHC damage [80, 106, 107]. Tbx18 is a critical TF for cell proliferation and cell fate determination, and it is also a target gene of the Hippo pathway [108].…”

Human induced pluripotent stem cells and CRISPR/Cas-mediated targeted genome editing: Platforms to tackle sensorineural hearing loss

“…[210][211][212] As a platform for modeling and understanding hearing disorders, GE and hiPSCs provide an attractive strategy to improve cell treatments of SHL (Figure 4A). For this purpose, hiPSCs carrying different deafness-causing mutations, such as in MYOSIN VIIA, 96 MYOSIN XVA, 95 the anion exchanger pendrin, 213 or A8344G of mitochondrial DNA (mtDNA), 214 have been used. After differentiation, the derived HC-like cells showed abnormal morphology and dysfunction, confirming the role and effects of investigated mutations.…”

“…214 After genetic correction (Figure 4B) utilizing CRISPR/Cas9 technology 95 or lentiviral transduction, 214 the morphology and function of hiPSC-derived HC-like cells were rescued. Specifically, the SB were more mature, 95,214 had restored organization, and similar electrophysiological function to hiPSC-derived HC-like cells from a healthy donor. 96 Interestingly, when hiPSCs obtained from a patient with Charcot-Marie-Tooth (CMT) disease type 1A (hipSC-Charcot-Marie-Tooth disease type 1A [CMT1A] 177 ) were cultured under the same conditions as the control line, hiPSC-CMT1A rarely generated SchCs (Table 1) through differentiation of NCCSCs.…”