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. 2024 Jul;57(7):e13620.
doi: 10.1111/cpr.13620. Epub 2024 Feb 24.

AAV-mediated Gpm6b expression supports hair cell reprogramming

Qiuhan Sun  1 , Liyan Zhang  1 , Tian Chen  1 , Nianci Li  1 , Fangzhi Tan  1 , Xingliang Gu  1 , Yinyi Zhou  1 , Ziyu Zhang  1 , Yicheng Lu  1 , Jie Lu  2 , Xiaoyun Qian  3 , Bing Guan  2 , Jieyu Qi  1 , Fanglei Ye  4 , Renjie Chai  1   5   6   7   8
Affiliations

AAV-mediated Gpm6b expression supports hair cell reprogramming

Qiuhan Sun et al. Cell Prolif. 2024 Jul.

Abstract

Irreversible damage to hair cells (HCs) in the cochlea leads to hearing loss. Cochlear supporting cells (SCs) in the murine cochlea have the potential to differentiate into HCs. Neuron membrane glycoprotein M6B (Gpm6b) as a four-transmembrane protein is a potential regulator of HC regeneration according to our previous research. In this study, we found that AAV-ie-mediated Gpm6b overexpression promoted SC-derived organoid expansion. Enhanced Gpm6b prevented the normal decrease in SC plasticity as the cochlea develops by supporting cells re-entry cell cycle and facilitating the SC-to-HC transformation. Also, overexpression of Gpm6b in the organ of Corti through the round window membrane injection facilitated the trans-differentiation of Lgr5+ SCs into HCs. In conclusion, our results suggest that Gpm6b overexpression promotes HC regeneration and highlights a promising target for hearing repair using the inner ear stem cells combined with AAV.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

FIGURE 1
FIGURE 1
Gpm6b overexpression promoted the proliferation of cochlear organoids. (A) Experimental design of the expansion culture of SCs in three‐dimensional in vitro assay. Given a single EdU treatment for 1 h on day 10. (B) Bright‐field images of cochlear organoids after expansion with the administration of AAV‐mNeonGreen (Control) and AAV‐Gpm6b, respectively. Scale bar: 50 μm. (C and D) The number (C) and diameter (D) of the organoids in (B). (E) Immunofluorescence images of the organoids in (B). Sox2 (red) marks SCs. EdU (cyan) marks proliferating cells. AAV‐mNeonGreen (green) marks cells transduced by AAVs. Scale bar: 50 μm. (F and G) The ratio of EdU+ organoids (F) and the ratio of EdU+ cells per organoid (G) in (E). (H–L) Similar analyses to (A to G) of cochlear organoids from P4 mice. Scale bars: 100 μm. AAV dose: 2 × 1010 GCs per well. Data are displayed as the mean with SEM. The value of p was calculated by Student's t‐test. *p < 0.05; ****p < 0.0001; n.s., no significance.
FIGURE 2
FIGURE 2
Gpm6b overexpression promoted HC regeneration in cochlear organoids. (A) Experimental design of the differentiation culture of cochlear organoids in the three‐dimensional in vitro assay. AAV dose: 2 × 1010 GCs per well. (B) Relative mRNA expression of Gpm6b in the cochlear organoids. (C) Immunofluorescence images of the organoids in (A). Scale bar: 100 μm. (D) The Myo7a+ organoids ratio was calculated in (C). (E–I) RNA sequencing data from cochlear organoids was added with control and AAV‐Gpm6b, respectively. (E) Correlation analysis between control and AAV‐Gpm6b–transduced organoids. (F) Venn diagram analysis of differentially expressed genes. (G) The volcano plot shows the overall profile of differentially expressed genes. Red/green dots indicate up/down‐regulated genes, respectively. Genes with no statistically significant expression difference are shown as blue dots. |log2FoldChange| > 1 and p‐value < 0.05 were the cutoffs for differentially expressed genes. (H) Heatmap of the differentially expressed genes. (I) GO terms significantly enriched in differentially expressed genes in Gpm6b‐overexpressing organoids. (J) The differentially expressed genes identified in (H) were verified by qPCR. Data are displayed as the mean with SEM. The value of p was calculated by Student's t‐test. **p < 0.01; ***p < 0.001; n.s. refers to no significance.
FIGURE 3
FIGURE 3
Gpm6b overexpression promoted hair cell regeneration in vivo. (A) Experimental design. (B) mRNA expression level of Gpm6b in P15 cochleae after the injection of AAV‐Gpm6b. (C) EdU immunostaining after Gpm6b overexpression in cochlear. EdU (cyan) marks proliferating cells. Sox2 (red) marks the SCs. Scale bar: 50 μm. (D) mRNA expression level of Lgr5 in P15 cochleae after the injection of AAV‐Gpm6b. (E) Immunofluorescence images of the AAV‐transduced cochleae of Lgr5‐EGFP mice. Lgr5‐EGFP (green) marks the Lgr5+ cells. (F) Experimental design. Tamoxifen was injected intraperitoneally into P1 wildtype mice. The AAVs were injected through the round window membrane, and the cochleae were collected at P7. (G) Immunofluorescence images of cochlear epithelia infected by control and AAV‐Gpm6b, respectively. Ectopic HCs are marked by yellow arrows (IHC area) and green arrows (OHC area). Scale bar: 50 μm. (H) The number of ectopic IHCs, ectopic OHCs, and total HCs in (B). AAV dose: 2.8 × 1010 GCs per cochlea. Data are displayed as the mean with SEM. The value of p was calculated by Student's t‐test. *p < 0.05; **p < 0.01; ***p < 0.001; n.s. refers to no significance.
FIGURE 4
FIGURE 4
Gpm6b overexpression facilitated the trans‐differentiation of Lgr5+ progenitors into HCs. (A) Experimental design. P1 Lgr5‐EGFPCreER/+/Rosa26‐tdTomatoloxp/+ mice were injected with tamoxifen intraperitoneally. AAVs were injected 0.5 days later through the round window membrane, and the cochleae were collected at P7. (B) Immunofluorescence of AAV‐transduced cochleae. Scale bar: 50 μm. (C) The number of Myo7a+/Tomato+ OHCs, IHCs and HCs per cochlea. AAV dose: 2.8 × 1010 GCs per cochlea. Data are displayed as the mean with SEM. The value of p was calculated by Student's t‐test. *p < 0.05; **p < 0.01; n.s. refers to no significance.
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