WNT Activation and TGFβ-Smad Inhibition Potentiate Stemness of Mammalian Auditory Neuroprogenitors for High-Throughput Generation of Functional Auditory Neurons In Vitro
Abstract
:1. Introduction
2. Materials and Methods
2.1. Spiral Ganglion Cells Isolation, In Vitro Culture and Differentiation
2.2. Cell Counting
2.3. Automated Analysis of ANPGs Neurosphere Growth
2.4. Cell Cycle Analysis by Flow Cytometry (FACS)
2.5. RNA Sequencing
2.6. Video Time Lapse Microscopy
2.7. Immunofluorescence and Confocal Microscopy
2.8. Electrophysiological Characterization of Differentiated SGNs
2.9. Statistical Analysis
3. Results
3.1. Intrinsic Stemness Properties of ANPGs Isolated from C57Bl/6 and A/J Mice
3.2. Comparative Transcriptomic Analysis (RNA-Sequencing) of High- vs. Low Stemness Neuroprogenitors
3.3. Pathway Enrichment Analysis and Differentially Represented Gene Ontologies between High and Low Stemness ANPGs
3.4. Reprogramming Stemness of Presenescent ANPGs
3.5. Phenotype of Stemness-Induced ANPGs (si-ANPGs)
3.6. Electrophysiological Characterization of Stemness-Induced Auditory Neurons (si-ANs) Differentiated from si-ANPGs
4. Discussion
5. Patents
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Cell Type | Phoenix | si-ANs | Primary SGN | Literature 1°SGN | ||
---|---|---|---|---|---|---|
Type 1 | Type 2 | Type 1 | Type 1 | Type 2 | ||
Abundance | 91% | 9% | 100% | 90–95% | 5–10% | [28] |
Vm (mV) | −40.9 ± 1.5 | −43.3 ± 1.5 | −40.2 ± 1.9 | −60.5 ± 0.9 | −65.3 ± 1.7 | [30] |
R (input) (MΩ) | 438 ± 39 | 233 ± 22 | 496 ± 87 | apex ~470 300 MΩ 510 ± 70 | Basal ~280 200 MΩ 360 ± 120 | [31] [28] [32] |
Spike latency (ms) | 3.1 ± 0.4 | 2.1 ± 0.1 | 4.4 ± 0.8 | 4.2 ± 0.8 | 4.8 ± 0.5 | [32] |
AP amplitude (mV) | 34.3 ± 10.7 | 49.2 ± 11.6 | 29.0 ± 5.4 | 50–95 | [33] | |
AP width (ms) | 2.2 ± 0.2 | 1.3 ± 0.2 | 2.5 ± 0.1 | Apex: 1.9 ± 0.01 Base: 1.68 ± 0.2 | Apex: 1.4 ± 0.02 Base: 1.25 ± 0.03 | [28] |
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Rousset, F.; Schilardi, G.; Sgroi, S.; Nacher-Soler, G.; Sipione, R.; Kleinlogel, S.; Senn, P. WNT Activation and TGFβ-Smad Inhibition Potentiate Stemness of Mammalian Auditory Neuroprogenitors for High-Throughput Generation of Functional Auditory Neurons In Vitro. Cells 2022, 11, 2431. https://doi.org/10.3390/cells11152431
Rousset F, Schilardi G, Sgroi S, Nacher-Soler G, Sipione R, Kleinlogel S, Senn P. WNT Activation and TGFβ-Smad Inhibition Potentiate Stemness of Mammalian Auditory Neuroprogenitors for High-Throughput Generation of Functional Auditory Neurons In Vitro. Cells. 2022; 11(15):2431. https://doi.org/10.3390/cells11152431
Chicago/Turabian StyleRousset, Francis, Giulia Schilardi, Stéphanie Sgroi, German Nacher-Soler, Rebecca Sipione, Sonja Kleinlogel, and Pascal Senn. 2022. "WNT Activation and TGFβ-Smad Inhibition Potentiate Stemness of Mammalian Auditory Neuroprogenitors for High-Throughput Generation of Functional Auditory Neurons In Vitro" Cells 11, no. 15: 2431. https://doi.org/10.3390/cells11152431
APA StyleRousset, F., Schilardi, G., Sgroi, S., Nacher-Soler, G., Sipione, R., Kleinlogel, S., & Senn, P. (2022). WNT Activation and TGFβ-Smad Inhibition Potentiate Stemness of Mammalian Auditory Neuroprogenitors for High-Throughput Generation of Functional Auditory Neurons In Vitro. Cells, 11(15), 2431. https://doi.org/10.3390/cells11152431