Cotransfection of Pax2 and Math1 promote in situ cochlear hair cell regeneration after neomycin insult

Abstract

The ideal strategy for hair cell regeneration is promoting residual supporting cell proliferation followed by induction of hair cell differentiation. In this study, cultured neonatal mouse organs of Corti were treated with neomycin to eliminate hair cells followed by incubation with recombined adenovirus expressing Pax2 and/or Math1. Results showed that overexpression of Pax2 significantly promoted proliferation of supporting cells. The number of BrdU⁺/myosin VIIA⁺ cells increased significantly in hair cell pre-existing region two weeks after adenovirus infection in Ad-Pax2-IRES-Math1 group compared to Ad-Pax2 and Ad-Math1 groups. This indicated that cotransfection of Pax2 and Math1 induced supporting cells to proliferate and differentiate into hair cells in situ. Most new hair cells were labeled by FM1-43 suggesting they acquired certain function. The results also suggest that inducing proliferating cells rather than quiescent cells to differentiate into hair cells by forced expression of Math1 is feasible for mammalian hair cell regeneration.

Figure 1. The high efficiency of gene expression mediated by recombinant adenovirus in cultured neonatal cochlear explants.

(A) EGFP fluorescence micrograph of an intact cultured neonatal cochlear explant stained with fluorescent phalloidin. The three rows of outer HCs (OHC) and one row of inner HCs (IHC) can be clearly seen. Arrows in (A4) pointed to transfection positive inner and outer hair cells. Arrowheads in (A2) pointed to transfection positive supporting cells. (B) Image of the positive transfection of supporting cells in the basal turn of a neomycin-damaged cochlear explant. Arrowheads in (B4) pointed to transfection positive supporting cells. Scale bars: 10 μm.

Figure 2. Forced Pax2 overexpression stimulated proliferation of supporting cells.

(A–D) Double immunofluorescence of BrdU and Sox2 in neomycin-damaged cochlear epithelium from the Ad-Empty, Ad-Pax2, Ad-Math1, and Ad-Pax2-IRES-Math1 groups. (E–H) Double immunofluorescence of BrdU and Sox2 in non-damaged cochlear explants from the Ad-Empty, Ad-Pax2, Ad-Math1, and Ad-Pax2-IRES-Math1 groups. Statistical data (I–K) showed that Pax2 overexpression increased the number of BrdU⁺, Sox2⁺, and BrdU⁺/Sox2⁺ cells. * (red): p < 0.05 vs. Ctr-E; ^& (red): p < 0.05 vs. Ctr-M; *(black): p < 0.05 vs. Neo-E; ^& (black): p < 0.05 vs. Neo-M. (L) The distribution of BrdU⁺ and BrdU⁺/Sox2⁺ cells in different regions throughout the damaged cochlea. Scale bars: 20 μm.

Figure 3. Forced expression of Pax2 stimulated cell proliferation.

(A and B) Double immunofluorescence of Pax2 and BrdU showed that most of Pax2 positive cells were proliferating in the neomycin-damaged cochlear epithelium in Ad-Pax2-IRES-Math1 group. Statistical data (E–F) showed that the number of Pax2⁺ and BrdU⁺/Pax2⁺ cells increased in Ad-Pax2 and Ad-Pax2-IRES-Math1 groups. *: p < 0.05 vs. Neo-E; ^&: p < 0.05 vs. Neo-M. (C) Double labeling of EGFP and BrdU in damaged cochlear epithelium at 7 days after Ad-Pax2-IRES-EGFP infection. (D) Higher magnification image of (C). Arrows pointed to proliferating supporting cells beneath pre-existing hair cells. The arrowhead pointed to a proliferating fibroblast cell. Scale bars: 20 μm in (A, B, and D); 50 μm in (C).

Figure 4. Forced Math1 expression promoted hair cell formation in the lesser epithelial ridge (LER).

(A–D) Double immunofluorescence of BrdU and myosin VIIA in the LER of neomycin-damaged cochlear epithelium at 2 weeks after adenovirus incubation. Myosin VIIA was used as a marker of hair cells. The arrows pointed to BrdU⁺/myosin VIIA⁺ hair cells that means hair cells formed by mitotic regeneration. The arrowheads pointed to BrdU⁻/myosin VIIA⁺ cells that mean hair cells formed by direct transdifferentiation in the LER. Statistical data (E–F) showed that Math1 expression promoted hair cell formation in the LER in both Ad-Math1 and Ad-Pax2-IRES-Math1 groups. *: p < 0.05 vs. Neo-E; ^#: p < 0.05 vs. Neo-P; ^&: p < 0.05 vs. Neo-M. Data in (G) showed the number of BrdU⁺/myosin VIIA⁺, BrdU⁻/myosin VIIA⁺, and total myosin VIIA⁺ cells in the LER at different times after adenovirus incubation. Scale bars: 20 μm.

Figure 5. Cotransfection of Pax2 and Math1 promoted in situ hair cell regeneration in neomycin-damaged cochleae.

(A–D) Double immunofluorescence of BrdU and myosin VIIA in hair cell pre-existing region (HCR) of neomycin-damaged cochlear epithelium at 2 weeks after adenovirus incubation. New hair cells were occasionally observed in Ad-empty (A, apical-middle turn), Ad-Pax2 (B, middle turn), and Ad-Math1 (C, basal turn) groups. However, in Ad-Pax2-IRES-Math1 group (D, basal turn), many BrdU⁺/myosin VIIA⁺ cells (arrows) were observed in HCR. Because hair cells in the cochlear basal turn are more sensitive to aminoglycoside antibiotics than those in the apical turn, more residual hair cells were observed in the apical turn than the basal turn after neomycin treatment. Arrows pointed to BrdU⁺/myosin VIIA⁺ cells. Statistical data (E–F) showed that the numbers of BrdU⁺/myosin VIIA⁺ and myosin VIIA⁺ cells significantly increased in Ad-Pax2-IRES-Math1 group, compared with other groups. *: p < 0.05 vs. Neo-E; ^#: p < 0.05 vs. Neo-P; ^&: p < 0.05 vs. Neo-M. Data in (G and J) showed the distribution of BrdU⁺/myosin VIIA⁺ and myosin VIIA⁺ cells in different regions throughout the damaged cochlea. Data in (H and I) showed the number of BrdU⁺/myosin VIIA⁺ and total myosin VIIA⁺ cells in the HCR at different times after adenovirus incubation. Scale bars: 10 μm.

Figure 6. Newly generated hair cells induced by co-expression of Pax2 and Math1 acquired certain function.

(A and B) Triple immunofluorescence of BrdU, Pax2, and myosin VIIA showed that the newly generated hair cells originated from transfection positive cells. Arrows pointed to BrdU⁺/myosin VIIA⁺/Pax2⁺ cells. (C) Double immunofluorescence of BrdU and FM1-43 suggested that newly generated hair cells developed mechanotransduction channels and acquired certain function. Arrow pointed to BrdU⁺/FM1-43⁺ cell. Data in (D) showed the number of BrdU⁺/FM1-43⁺ cells in the HCR and LER at 2 weeks after adenovirus incubation. (E) Triple immunofluorescence of BrdU, myosin VIIA, and FM1-43 confirmed that the newly generated hair cells acquired certain function. Arrow pointed to BrdU⁺/myosin VIIA⁺/FM1-43⁺ cell. Scale bars: 5 μm in (A and C); 10 μm in (B and E).