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. 2023 Apr 17;120(17):e2215253120. doi: 10.1073/pnas.2215253120

Fig. 4.

Fig. 4.

A cocktail of siRNAs and small molecules to replace MYC/NICD in reprogramming for HC-like cell regeneration in adult cochlea in vitro. (A) A schematic diagram illustrating the experimental procedure of VLFsiFsiM/Ad.Atoh1 induced HC-like cell regeneration. (B and C) The cocktail (VLFsiFsiM)/Ad.Atoh1.mCherry or vehicle/Ad.Atoh1.mCherry treated adult (P30) WT mouse cochlear samples were labeled with MYO7A (blue)/POU4F3 (green) in the sensory epithelial region. Numerous HC-like cells (POU4F3+/MYO7A+) were seen in the apex of the VLFsiFsiM/Ad.Atoh1.mCherry-treated cochlea. (D–G) Quantification and comparison of infected cells and regenerated HC-like cells in the apical turn of the cultured cochleae between VLFsiFsiM/Ad.Atoh1.mCherry-treated and vehicle/Ad.Atoh1.mCherry-treated groups. Significantly more HC-like cells were regenerated in the VLFsiFsiM/Ad.Atoh1-treated than vehicle/Ad.Atoh1-treated samples (E and G) in the sensory epithelial (SE) and the limbus (Lib) regions, respectively. The number of Ad.Atoh1.mCherry-infected cells between the two conditions was similar (D and F). (H) A schematic diagram illustrating the experimental procedure of lineage tracing for new HC-like cells after VLFsiFsiM/Ad.Atoh1 treatment. (I) In the cultured Sox2CreER/tdT adult cochlea, Tamoxifen (Tamo) induced tdT in the SCs which transdifferentiated to HC-like cells by VLFsiFsiM/Ad.Atoh1 treatment, shown by MYO7A+/Sox2-tdT+ labeling. The arrow indicates an existing HC, which was MYO7A+/Sox2-tdT−. The arrowhead indicates a regenerated HC-like cell, shown by MYO7A+/Sox2-tdT+ labeling. (J) Enlarge inset from H to show the lineage traced HC-like cells (MYO7A+/Sox2-tdT+) (arrowheads). (K) VLFsiFsiM/Ad.Atoh1-treated adult (P30) Atoh1-GFP mouse cochlear samples showed infected cells (GFP+) that transdifferentiated to HC-like cell (MYO7A+) and were able to take up FM1-43. (L) A cluster of HC-like cells (MYO7A+) was seen in the VLFsiFsiM/Ad.Atoh1-treated adult cochlea. T, Tamo: 4-hydroxytamoxifen. V: VPA; L: LiCl; F: FSK; siF: siFIR; siM: siMxi1. SE: sensory epithelial region. Lib: Limbus Region. **P < 0.01, ***P < 0.001, Student’s t test. Error bar, mean ± SEM; N = 5 to 6 in each group. Source data are provided as a Source Data file. (Scale bars, 10 μm.)