modeling-mitochondrial-disease-using-brain-organoids-focus-on

Modeling Mitochondrial Disease Using Brain Organoids: A Focus on Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like Episodes

Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes (MELAS) are mitochondrial disorders most commonly caused by a m.3243A&#62;G ...

Kitasato University School of Medicine

Kitasato University Graduate School of Medical Science

Efficient induction of the otic placode, the developmental origin of the inner ear from human pluripotent stem cells (hPSCs), provides a robust platform for otic development and sensorineural hearing loss modelling. Nevertheless, there remains a limited capacity of otic lineage specification from hPSCs by stepwise differentiation methods, since the critical factors for successful otic cell differentiation have not been thoroughly investigated. In this study, we developed a novel differentiation system involving the use of a three-dimensional (3D) floating culture with signalling factors for generating otic cell lineages via stepwise differentiation of hPSCs.

Critical roles of FGF, RA, and WNT signalling in the development of the human otic placode and subsequent lineages in a dish.

We report the establishment of two human induced pluripotent stem cell (iPSC) lines from individuals without auditory disorders. Extensive audiometry tests were performed to confirm normal hearing. The generated iPSC lines expressed pluripotency genes and showed differentiation capability into the three germ layers. The iPSC lines will be used as controls for pathological analysis and drug screening for ear disorders.

Generation of two induced pluripotent stem cell lines from individuals without auditory disorders.

Otof, which encodes otoferlin, knockout mice are considered model mice for auditory neuropathy spectrum disorder, which is characterized by an absent auditory brainstem response (ABR) despite preserved distortion product otoacoustic emission (DPOAE). Although otoferlin-deficient mice lack neurotransmitter release at the inner hair cell (IHC) synapse, it remains unclear how the Otof mutation affects spiral ganglions. Thus, we used Otof-mutant mice carrying the Otof allele (Otof) and analyzed spiral ganglion neurons (SGNs) in Otof mice by immunolabeling type Ⅰ SGNs (SGN-Ⅰ) and type II SGNs (SGN-II). We also examined apoptotic cells in SGNs. Four-week-old Otof mice had an absent ABR but normal DPOAEs. The number of SGNs was significantly lower in Otof mice on postnatal day 7 (P7), P14, and P28 compared with that of wild-type mice. Moreover, significantly more apoptotic SGNs were observed in Otof mice than in wild-type mice on P7, P14, and P28. SGN-IIs were not significantly reduced in Otof mice on P7, P14, and P28. No apoptotic SGN-IIs were observed under our experimental conditions. In summary, Otof mice showed a reduction in SGNs accompanied by apoptosis of SGN-Ⅰs even before the onset of hearing. We speculate that the reduction in SGNs with apoptosis is a secondary defect caused by a lack of otoferlin in IHCs. Appropriate glutamatergic synaptic inputs may be important for the survival of SGNs.

Apoptosis of type I spiral ganglion neuron cells in Otof-mutant mice.

Mammalian auditory hair cells transduce sound-evoked traveling waves in the cochlea into nerve stimuli, which are essential for hearing function. Pillar cells located between the inner and outer hair cells are involved in the formation of the tunnel of Corti, which incorporates outer-hair-cell-driven fluid oscillation and basilar membrane movement, leading to the fine-tuned frequency-specific perception of sounds by the inner hair cells. However, the detailed molecular mechanism underlying the development and maintenance of pillar cells remains to be elucidated. In this study, we examined the expression and function of brain-specific angiogenesis inhibitor 3 (Bai3), an adhesion G-protein-coupled receptor, in the cochlea. We found that Bai3 was expressed in hair cells in neonatal mice and pillar cells in adult mice, and, interestingly,  knockout mice revealed the abnormal formation of pillar cells, with the elevation of the hearing threshold in a frequency-dependent manner. Furthermore, old  knockout mice showed the degeneration of hair cells and spiral ganglion neurons in the basal turn. The results suggest that Bai3 plays a crucial role in the development and/or maintenance of pillar cells, which, in turn, are necessary for normal hearing function. Our results may contribute to understanding the mechanisms of hearing loss in human patients.

Brain-Specific Angiogenesis Inhibitor 3 Is Expressed in the Cochlea and Is Necessary for Hearing Function in Mice.

We report the establishment of a human induced pluripotent stem cell (iPSC) line from a 54-year-old male patient with an A1555G mutation in the mitochondrial 12S ribosomal RNA gene (MTRNR1), associated with sensorineural hearing loss. The established iPSC line expressed stemness markers or undifferentiated state markers. We also demonstrated the capacity of the cells to differentiate into the three germ layers, suggesting its pluripotency and utility in the pathological study of sensorineural hearing loss and drug screening for ear disorders.

Generation of an induced pluripotent stem cell line (KEIUi008-A) from a hearing loss patient with an A1555G mutation in mitochondrial DNA.

Disease-related cells differentiated from patient-derived iPSCs are useful for elucidating the pathophysiological mechanisms underlying these diseases. In this study, four iPSC lines were established from independent patients with sensorineural hearing loss and a mutation in EYA4. These iPSCs showed pluripotency, the capacity to differentiate into three germ layers, and normal karyotypes, suggesting that these lines are useful for the pathological study of sensorineural hearing loss and drug screening for ear disorders.

Generation of four induced pluripotent stem cell lines (KEIUi004-A, KEIUi005-A, KEIUi006-A, and KEIUi007-A) from patients with sensorineural hearing loss with mutation in EYA4 gene.

Article	Year
Critical roles of FGF, RA, and WNT signalling in the development of the human otic placode and subsequent lineages in a dish. Regenerative therapy\| PubMed ID: 35620640
Generation of two induced pluripotent stem cell lines from individuals without auditory disorders. Stem cell research\| PubMed ID: 36630839	2023
Apoptosis of type I spiral ganglion neuron cells in Otof-mutant mice. Neuroscience letters\| PubMed ID: 36914046	2023
Brain-Specific Angiogenesis Inhibitor 3 Is Expressed in the Cochlea and Is Necessary for Hearing Function in Mice. International journal of molecular sciences\| PubMed ID: 38069416	2023
Generation of an induced pluripotent stem cell line (KEIUi008-A) from a hearing loss patient with an A1555G mutation in mitochondrial DNA. Stem cell research\| PubMed ID: 38815527	2024
Generation of four induced pluripotent stem cell lines (KEIUi004-A, KEIUi005-A, KEIUi006-A, and KEIUi007-A) from patients with sensorineural hearing loss with mutation in EYA4 gene. Stem cell research\| PubMed ID: 39002249	2024

Chika Saegusa

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