anti math 1  (Bioss)

Journal: Advanced Science

Article Title: AAV‐mediated Gene Cocktails Enhance Supporting Cell Reprogramming and Hair Cell Regeneration

doi: 10.1002/advs.202304551

Figure Lengend Snippet: AAV‐ie mediated Atoh1, GPA, and GPAS effectively regenerated HC‐like cells in vivo. A) Strategy for the overexpression of exogenous Atoh1 (A), Atoh1‐Six1 (AS), and Gfi‐Pou4f3 (GP) mediated by AAV‐ie in the neonatal mouse inner ear. The P2A polypeptide sequence mediates protein self‐splicing after translation. All the proteins were tagged with an HA (hemagglutinin) tag. B) Experimental design diagram. All the AAVs were delivered to the mice's left ear at dose:2.5E9 GCs per ear, then the cochleae were obtained at P15. C) The qPCR showing the relative mRNA expression of Atoh1 , Gfi1 , Pou4f3 , and Six1 in the cochleae injected with AAV‐control, AAV‐ Atoh1 , AAV‐ Six1 , AAV‐GPA, and AAV‐GPAS. Raw results from 3 replicated experiments. Error bars are ±SEM. ** p < 0.01, *** p < 0.001. D) Representative immunoblotting images of Atoh1, Gfi1, Pou4f3, and Six1 in the cochleae injected with AAV‐control, AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS. 4 repeated experiments, and 1 representative images. E) The western blotting images of Atoh1, Gfi1, Pou4f3, and Six1 in the left ear were injected with AAV‐GPAS. Six1 antibody was used for the immunoprecipitation, and western blotting was used to analyze the co‐expressed proteins using Atoh1, Pou4f3, or Gfi1 antibody. 4 repeated experiments, and 1 representative image. F) Representative confocal images of Myosin7a (red, HC marker) signals in the basal, middle, and apical turns of the ear injected with AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS. All the AAVs were delivered to the mice's left ear at a dose of 2.5E9 GCs per ear. Scale bar, 50 µm. G) The number of Myosin7a in the AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS injected cochlea in (F). N = 5, error bars are ±SEM, **** p < 0.0001. H) Representative confocal images of Myosin7a (red, HC marker) signals in the apical turns of the ear injected with AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS. All the AAVs were delivered to the mice's left ear, at a dose of 5.25E9 GCs per ear. Scale bar, 50 µm.

Article Snippet: The primary antibodies were Atoh1 (1:1000 dilution, Santa Cruz, sc‐136173), Pou4f3 (1:1000 dilution, Santa Cruz, sc‐81980), Gfi1 (1:1000 dilution, Santa Cruz, sc‐373960), Six1 (1:1000 dilution, Sigma, HPA001893), and GAPDH (1:2000 dilution, Abcam, ab8245).

Techniques: In Vivo, Over Expression, Sequencing, Expressing, Injection, Control, Western Blot, Immunoprecipitation, Marker

Journal: Advanced Science

Article Title: AAV‐mediated Gene Cocktails Enhance Supporting Cell Reprogramming and Hair Cell Regeneration

doi: 10.1002/advs.202304551

Figure Lengend Snippet: Single‐nucleus transcriptomic analysis and cell trajectory analysis of AAV‐induced HCs. A) UMAP plot showing seven potential subtypes HCs in the AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS groups. B) Heatmap illustrating shared and group‐specific differentially expressed genes in each subtype related to (A). C) Venn diagrams showing differentially expressed genes in each subtype from the AAV‐ Atoh1 (blue) and AAV‐GPA (violet) groups compared to the AAV‐GPAS group (orange). D) The proportions of HC subtypes from the 3 groups using the Seurat R package. E) The trajectory manifold of AAV‐infected HCs from the AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS groups using the Monocle 2 algorithm. Dotted and solid lines show the different cell fates /trajectories confirmed by expression profiles. F) Heatmap of significant genes according to branch expression analysis comparing the HC fates in the 3 groups. The genes in the heatmap were used to analyze the pseudotime‐based variation. G) Top: Illustration of the experimental design. AAVs were added to the culture medium, dose: 3E10 GCs per well. Bottom: The qPCR data showed the transcription‐level expression of Chd7 , Ankrd6 , Zeb1 , and Supervillin mRNA in the AAV‐Ctrl/Atoh1/GPA/GPAS overexpressing organoids. Raw results from 3 replicated experiments. Error bars are ±SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The primary antibodies were Atoh1 (1:1000 dilution, Santa Cruz, sc‐136173), Pou4f3 (1:1000 dilution, Santa Cruz, sc‐81980), Gfi1 (1:1000 dilution, Santa Cruz, sc‐373960), Six1 (1:1000 dilution, Sigma, HPA001893), and GAPDH (1:2000 dilution, Abcam, ab8245).

Techniques: Infection, Expressing

Journal: Advanced Science

Article Title: AAV‐mediated Gene Cocktails Enhance Supporting Cell Reprogramming and Hair Cell Regeneration

doi: 10.1002/advs.202304551

Figure Lengend Snippet: AAV‐ie‐mediated GP and GPS overexpression enhanced the OHC‐like cell regeneration. A) Experimental design diagram. All the AAVs were delivered to the mice's left ear, at dose:2.5E9 GCs per ear, then the cochleae were obtained at P15. B) Representative confocal images of Myosin7a and Tomato signals in the apical turns of the ear injected with AAV‐GPAS. Red: Tomato, cyan: Myosin7a. Scale bars, 20 µm. C) The immunofluorescence images of Myosin7a and Tomato signals in the basal, middle, and apical turns of the ear injected with AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS. Magnified areas from the OHC region, Red: Tomato, cyan: Myosin7a. Scale bars, 20 µm. D) The number of Myosin7a (Myo7a) + /Tomato + cells in the ear transduced by AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS, corresponding to (C). N = 8, error bars are ±SEM. * p < 0.05. E) Representative confocal images of Slc26a5 (an OHC marker) and Tomato signals in the basal, middle, and apical turns of the ear injected with AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS. Arrows point to Slc26a5 + / Tomato + regenerated HCs. Red: Tomato, cyan: Slc26a5. Scale bars, 20 µm. F) The number of double‐positive Slc26a5 + /Tomato + cells in the ear transduced by AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS correspond to (E). N = 8, error bars are ±SEM. * p < 0.05. (G) The qPCR showing the Slc26a5 , Ikzf2 , and Insm1 mRNA expression in the ear transduced with AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS, respectively. Raw results from 3 replicated experiments. Error bars are ±SEM. * p < 0.05, *** p < 0.001.

Article Snippet: The primary antibodies were Atoh1 (1:1000 dilution, Santa Cruz, sc‐136173), Pou4f3 (1:1000 dilution, Santa Cruz, sc‐81980), Gfi1 (1:1000 dilution, Santa Cruz, sc‐373960), Six1 (1:1000 dilution, Sigma, HPA001893), and GAPDH (1:2000 dilution, Abcam, ab8245).

Techniques: Over Expression, Injection, Immunofluorescence, Marker, Expressing, Transduction

Journal: Advanced Science

Article Title: AAV‐mediated Gene Cocktails Enhance Supporting Cell Reprogramming and Hair Cell Regeneration

doi: 10.1002/advs.202304551

Figure Lengend Snippet: AAV‐ie‐mediated GPS overexpression strongly promoted the efficiency of Atoh1 ‐indued HC‐like cell regeneration in the IHC region. A) Experimental design diagram. All the AAVs were delivered to the mice's left ear, at dose:2.5E9 GCs per ear, then the cochleae were obtained at P15. B) The immunofluorescence images of Myosin7a and Tomato signals in the basal, middle, and apical turns of the ear injected with AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS. Magnified areas from the IHC region corresponding to (Figure ), Red: Tomato, cyan: Myosin7a. Scale bars, 20 µm. C) The number of double‐positive Myo7a + /Tomato + cells in the ear transduced by AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS correspond to (B). N = 8, error bars are ±SEM. **** p < 0.0001. D) Representative confocal images of Myosin7a and Tomato signals in the basal, middle, and apical turns of the ear injected with AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS. Magnified areas from the GER region corresponding to (Figure ), Red: Tomato, cyan: Myosin7a. Scale bars, 20 µm. E) The number of double‐positive Myo7a + /Tomato + cells in the ear transduced by AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS, respectively, corresponds to (C). N = 8, error bars are ±SEM. ** p < 0.01, **** p < 0.0001. F) Representative confocal images of Myosin7a and Otoferlin (an IHC marker) signals in the apical turns of cochleae injected with AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS. Red: Myosin7a, cyan: Otoferlin. Scale bars, 50 µm. G) The qPCR showing the Otof , Vglut3 , and Tbx2 relative mRNA expression in the ear transduced with AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS. Raw results from 3 replicated experiments, error bars are ±SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies were Atoh1 (1:1000 dilution, Santa Cruz, sc‐136173), Pou4f3 (1:1000 dilution, Santa Cruz, sc‐81980), Gfi1 (1:1000 dilution, Santa Cruz, sc‐373960), Six1 (1:1000 dilution, Sigma, HPA001893), and GAPDH (1:2000 dilution, Abcam, ab8245).

Techniques: Over Expression, Immunofluorescence, Injection, Marker, Expressing, Transduction

Journal: Advanced Science

Article Title: AAV‐mediated Gene Cocktails Enhance Supporting Cell Reprogramming and Hair Cell Regeneration

doi: 10.1002/advs.202304551

Figure Lengend Snippet: AAV‐ie‐mediated GPS overexpression enhanced the maturation of Atoh1 ‐mediated HC‐like cell regeneration in vivo. A) Experimental design diagram. All the AAVs were delivered to the mice's left ear, dose: 2.5E9 GCs per ear, then the cochleae were obtained at P15. B) The immunofluorescence images of FM1‐43 and Myosin7a signals in the basal, middle, and apical turns of the ear from P15 mice injected with AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS. Red: FM1‐43, cyan: Myosin7a. Scale bar, 40 µm. C) The signal intensity of FM1‐43 in ear transduced by AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS, respectively, corresponding to (B). Raw results from 3 replicated experiments, error bars are ±SEM. **** p < 0.0001. D) Example membrane potentials of native HCs and Atoh1‐OE (overexpression), GPA‐OE, and GPAS‐OE HC‐like cells from P15 mice recorded in a current‐clamp mode. The initial calcium spike is plotted as a red line and highlighted with a red arrow. The confocal images of HC‐like cells after electrophysiological recording in the AAV‐GPAS transduced cochlea. Red: Myosin7a, HCs marker, cyan: Strepavidin, the marker of electrophysiological recording HC‐like cells. E) Dot‐plot showing the resting membrane potential (RMP) recorded in cells from each group corresponding with (D). A descending gradient in RMP was observed from the Atoh1‐OE to GPAS‐OE group that was in parallel with an ascending gradient in cell maturity. N = 5, error bars are ±SEM. ** p < 0.01, **** p < 0.0001, n.s., not significant. F) Dot‐plot showing the individual and mean values of injected currents that evoked the initial spike recorded in cells from the 4 groups. N = 5, error bars are ±SEM. ** p < 0.01, **** p < 0.0001. G) Example traces of spontaneous membrane potentials from native IHCs and Atoh1‐OE and GPAS‐OE HC‐like cells. A calcium‐induced burst‐spiking pattern was evident with over‐expression of Atoh1 alone, but this was abolished in both native HCs and GPAS‐OE cells.

Article Snippet: The primary antibodies were Atoh1 (1:1000 dilution, Santa Cruz, sc‐136173), Pou4f3 (1:1000 dilution, Santa Cruz, sc‐81980), Gfi1 (1:1000 dilution, Santa Cruz, sc‐373960), Six1 (1:1000 dilution, Sigma, HPA001893), and GAPDH (1:2000 dilution, Abcam, ab8245).

Techniques: Over Expression, In Vivo, Immunofluorescence, Injection, Membrane, Marker

Journal: Advanced Science

Article Title: AAV‐mediated Gene Cocktails Enhance Supporting Cell Reprogramming and Hair Cell Regeneration

doi: 10.1002/advs.202304551

Figure Lengend Snippet: AAV‐ie‐mediated Atoh1, GPA, and GPAS effectively regenerated HC‐like cells in P7 mice. A) Experimental design diagram. All the AAVs were delivered to the mice's left ear, dose:2.5E9 GCs per ear. B) The qPCR showing the relative mRNA expression of Atoh1 , Gfi1 , Pou4f3 , and Six1 in the cochleae injected with AAV‐control, AAV‐ Atoh1 , AAV‐ Six1 , AAV‐GPA, and AAV‐GPAS. Raw results from 3 replicated experiments. Error bars are ±SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. C) The immunofluorescence images of Myosin7a signals in the basal and middle turns of the ear injected with AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS, respectively at P16 mice. Red: Myosin7a. Scale bar, 30 µm. D) The number of Myosin7a in the AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS injected ear in (C). N = 5, error bars are ±SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. E) The immunofluorescence images of Myosin7a and Otoferlin signals in the basal and middle turns of the ear injected with AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS, at P16 mice, respectively. Magnified areas corresponding to (C). Red: Myosin7a, cyan: Otoferlin. Scale bar, 30 µm. F) The percentage of the ectopic double‐positive IHC + /Otoferlin + cells in the cochleae corresponding to (E). N = 5, error bars are ±SEM. * p < 0.05, ** p < 0.01. G) Representative confocal images of Myosin7a and Phalloidin signals in the apical to middle turns of the ear injected with AAV‐ Atoh1 , AAV‐GPA, and AAV‐GPAS, respectively, at P16 mice. Red: Myosin7a, cyan: Phalloidin. Scale bar, 30 µm. H) Representative confocal images of Myosin7a, Sox2, and Myo15‐Green signals in the apical turn of ear injected with Anc80L65‐Myo15‐mNeonGreen at P2 WT mice. Red: Sox2, Magenta: Myosin7a. Scale bar, 200 µm. I) The ABR results of WT mice, AAV‐DTR (AAV‐Myo15‐DTR‐mNeonGreen), and AAV‐DTR+AAV‐GPAS injected ear at P30 mice. N = 4, error bars are ±SEM. J) Representative confocal images of Myosin7a and DAPI signals in the apical turn of the ear injected with AAV‐DTR at P30 mice. Red: Myosin7a, grey: DAPI. Scale bar, 200 µm. K) Representative confocal images of Myosin7a, HA, and DTR‐Green signals in the apical and basal turns of ear injected with AAV‐DTR+AAV‐GPAS at P30 mice. Red: Myosin7a, cyan: HA, Green: AAV‐DTR‐mNeoNGreen. Scale bar, 40 µm.

Article Snippet: The primary antibodies were Atoh1 (1:1000 dilution, Santa Cruz, sc‐136173), Pou4f3 (1:1000 dilution, Santa Cruz, sc‐81980), Gfi1 (1:1000 dilution, Santa Cruz, sc‐373960), Six1 (1:1000 dilution, Sigma, HPA001893), and GAPDH (1:2000 dilution, Abcam, ab8245).

Techniques: Expressing, Injection, Control, Immunofluorescence