WNT Activation and TGFβ-Smad Inhibition Potentiate Stemness of Mammalian Auditory Neuroprogenitors for High-Throughput Generation of Functional Auditory Neurons In Vitro
Hearing loss affects over 460 million people worldwide and is a major socioeconomic burden. Both genetic and environmental factors (i.e., noise overexposure, ototoxic drug treatment and ageing), promote the irreversible degeneration of cochlear hair cells and associated auditory neurons, leading to...
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MDPI AG
2022-08-01
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| author | Francis Rousset Giulia Schilardi Stéphanie Sgroi German Nacher-Soler Rebecca Sipione Sonja Kleinlogel Pascal Senn |
| author_facet | Francis Rousset Giulia Schilardi Stéphanie Sgroi German Nacher-Soler Rebecca Sipione Sonja Kleinlogel Pascal Senn |
| author_sort | Francis Rousset |
| collection | DOAJ |
| description | Hearing loss affects over 460 million people worldwide and is a major socioeconomic burden. Both genetic and environmental factors (i.e., noise overexposure, ototoxic drug treatment and ageing), promote the irreversible degeneration of cochlear hair cells and associated auditory neurons, leading to sensorineural hearing loss. In contrast to birds, fish and amphibians, the mammalian inner ear is virtually unable to regenerate due to the limited stemness of auditory progenitors, and no causal treatment is able to prevent or reverse hearing loss. As of today, a main limitation for the development of otoprotective or otoregenerative therapies is the lack of efficient preclinical models compatible with high-throughput screening of drug candidates. Currently, the research field mainly relies on primary organotypic inner ear cultures, resulting in high variability, low throughput, high associated costs and ethical concerns. We previously identified and characterized the phoenix auditory neuroprogenitors (ANPGs) as highly proliferative progenitor cells isolated from the A/J mouse cochlea. In the present study, we aim at identifying the signaling pathways responsible for the intrinsic high stemness of phoenix ANPGs. A transcriptomic comparison of traditionally low-stemness ANPGs, isolated from C57Bl/6 and A/J mice at early passages, and high-stemness phoenix ANPGs was performed, allowing the identification of several differentially expressed pathways. Based on differentially regulated pathways, we developed a reprogramming protocol to induce high stemness in presenescent ANPGs (i.e., from C57Bl6 mouse). The pharmacological combination of the WNT agonist (CHIR99021) and TGFβ/Smad inhibitors (LDN193189 and SB431542) resulted in a dramatic increase in presenescent neurosphere growth, and the possibility to expand ANPGs is virtually limitless. As with the phoenix ANPGs, stemness-induced ANPGs could be frozen and thawed, enabling distribution to other laboratories. Importantly, even after 20 passages, stemness-induced ANPGs retained their ability to differentiate into electrophysiologically mature type I auditory neurons. Both stemness-induced and phoenix ANPGs resolve a main bottleneck in the field, allowing efficient, high-throughput, low-cost and 3R-compatible in vitro screening of otoprotective and otoregenerative drug candidates. This study may also add new perspectives to the field of inner ear regeneration. |
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| spelling | doaj.art-c66d491cdd084cf2b4bc9e9456e542f02023-12-03T12:32:46ZengMDPI AGCells2073-44092022-08-011115243110.3390/cells11152431WNT Activation and TGFβ-Smad Inhibition Potentiate Stemness of Mammalian Auditory Neuroprogenitors for High-Throughput Generation of Functional Auditory Neurons In VitroFrancis Rousset0Giulia Schilardi1Stéphanie Sgroi2German Nacher-Soler3Rebecca Sipione4Sonja Kleinlogel5Pascal Senn6The Inner Ear and Olfaction Lab, Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, 1206 Geneva, SwitzerlandInstitute of Physiology, Department of Biomedical Research (DBMR), University of Bern, 3012 Bern, SwitzerlandThe Inner Ear and Olfaction Lab, Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, 1206 Geneva, SwitzerlandThe Inner Ear and Olfaction Lab, Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, 1206 Geneva, SwitzerlandThe Inner Ear and Olfaction Lab, Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, 1206 Geneva, SwitzerlandInstitute of Physiology, Department of Biomedical Research (DBMR), University of Bern, 3012 Bern, SwitzerlandThe Inner Ear and Olfaction Lab, Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, 1206 Geneva, SwitzerlandHearing loss affects over 460 million people worldwide and is a major socioeconomic burden. Both genetic and environmental factors (i.e., noise overexposure, ototoxic drug treatment and ageing), promote the irreversible degeneration of cochlear hair cells and associated auditory neurons, leading to sensorineural hearing loss. In contrast to birds, fish and amphibians, the mammalian inner ear is virtually unable to regenerate due to the limited stemness of auditory progenitors, and no causal treatment is able to prevent or reverse hearing loss. As of today, a main limitation for the development of otoprotective or otoregenerative therapies is the lack of efficient preclinical models compatible with high-throughput screening of drug candidates. Currently, the research field mainly relies on primary organotypic inner ear cultures, resulting in high variability, low throughput, high associated costs and ethical concerns. We previously identified and characterized the phoenix auditory neuroprogenitors (ANPGs) as highly proliferative progenitor cells isolated from the A/J mouse cochlea. In the present study, we aim at identifying the signaling pathways responsible for the intrinsic high stemness of phoenix ANPGs. A transcriptomic comparison of traditionally low-stemness ANPGs, isolated from C57Bl/6 and A/J mice at early passages, and high-stemness phoenix ANPGs was performed, allowing the identification of several differentially expressed pathways. Based on differentially regulated pathways, we developed a reprogramming protocol to induce high stemness in presenescent ANPGs (i.e., from C57Bl6 mouse). The pharmacological combination of the WNT agonist (CHIR99021) and TGFβ/Smad inhibitors (LDN193189 and SB431542) resulted in a dramatic increase in presenescent neurosphere growth, and the possibility to expand ANPGs is virtually limitless. As with the phoenix ANPGs, stemness-induced ANPGs could be frozen and thawed, enabling distribution to other laboratories. Importantly, even after 20 passages, stemness-induced ANPGs retained their ability to differentiate into electrophysiologically mature type I auditory neurons. Both stemness-induced and phoenix ANPGs resolve a main bottleneck in the field, allowing efficient, high-throughput, low-cost and 3R-compatible in vitro screening of otoprotective and otoregenerative drug candidates. This study may also add new perspectives to the field of inner ear regeneration.https://www.mdpi.com/2073-4409/11/15/2431otic progenitorsauditory neuroprogenitorsauditory neuronsauditory progenitors stemnessauditory neuron regenerationin vitro model |
| spellingShingle | Francis Rousset Giulia Schilardi Stéphanie Sgroi German Nacher-Soler Rebecca Sipione Sonja Kleinlogel Pascal Senn WNT Activation and TGFβ-Smad Inhibition Potentiate Stemness of Mammalian Auditory Neuroprogenitors for High-Throughput Generation of Functional Auditory Neurons In Vitro Cells otic progenitors auditory neuroprogenitors auditory neurons auditory progenitors stemness auditory neuron regeneration in vitro model |
| title | WNT Activation and TGFβ-Smad Inhibition Potentiate Stemness of Mammalian Auditory Neuroprogenitors for High-Throughput Generation of Functional Auditory Neurons In Vitro |
| title_full | WNT Activation and TGFβ-Smad Inhibition Potentiate Stemness of Mammalian Auditory Neuroprogenitors for High-Throughput Generation of Functional Auditory Neurons In Vitro |
| title_fullStr | WNT Activation and TGFβ-Smad Inhibition Potentiate Stemness of Mammalian Auditory Neuroprogenitors for High-Throughput Generation of Functional Auditory Neurons In Vitro |
| title_full_unstemmed | WNT Activation and TGFβ-Smad Inhibition Potentiate Stemness of Mammalian Auditory Neuroprogenitors for High-Throughput Generation of Functional Auditory Neurons In Vitro |
| title_short | WNT Activation and TGFβ-Smad Inhibition Potentiate Stemness of Mammalian Auditory Neuroprogenitors for High-Throughput Generation of Functional Auditory Neurons In Vitro |
| title_sort | wnt activation and tgfβ smad inhibition potentiate stemness of mammalian auditory neuroprogenitors for high throughput generation of functional auditory neurons in vitro |
| topic | otic progenitors auditory neuroprogenitors auditory neurons auditory progenitors stemness auditory neuron regeneration in vitro model |
| url | https://www.mdpi.com/2073-4409/11/15/2431 |
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