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. Author manuscript; available in PMC: 2021 May 1.
Published in final edited form as: Acta Biomater. 2020 Mar 7;108:111–127. doi: 10.1016/j.actbio.2020.03.007

Figure 9. (A):

Figure 9.

Immunohistochemistry of the DTR mouse cochlea. Confocal fluorescence imaged of phalloidin (red) and myosin VIIa (green) in the whole mounted auditory epithelium. (a) A wild type cochlea (control) shows a single row of IHCs and three rows of OHCs in the basal turn. (b) A DTR mouse cochlea without DT injection also shows a single row of IHCs and three rows of OHCs in the basal turn as well. (c and d) A DTR mouse cochlea with DT injection at P25 demonstrates no IHCs or OHCs in the basal turn (c) and the apical turn (d). OHC: outer hair cells; IHC: inner hair cells; PC: Pillar cells, DC: Deiters cells, and ISC: inner supporting cells. B): Immunohistochemistry of transplanted hESC-derived ONP spheroids in the DTR mouse cochlea. Left column: a corresponding low-power magnification microphotograph of the DTR mouse cochlea (10X). Each yellow circle indicates the anatomical location of a transplanted hESC-derived ONP spheroid. Right column: high-power magnification microphotographs of transplanted hESC-derived ONP spheroids stained with TOTO3 (nuclear counterstaining), β-III tubulin, and AHNA (40X). (a) Human ESC-derived ONP spheroids transplanted with 1% GrowDex®-T. (b) Human ESC-derived ONP spheroids transplanted with 1% GrowDex®-T and 800,000 of PODS®-hBDNF. Small white arrow: neurites. (c) Another hESC-derived ONP spheroid transplanted with 1% GrowDex®-T and 800,000 of PODS®-hBDNF. SM: scala media, SV: scala vestibuli, ST: scala tympani, MT: the middle turn of the cochlea, and MO: modiolus. (C): (a) Quantification of the number of ANHA positive cells (profile) in three different turns of the DTR mouse cochleae. (b) Quantification of the number of the ANHA positive cells (profile) in dissociated ONPs transplantation vs. ONP spheroids transplantation in four anatomical subdivisions of the cochlea. (c) Quantification of the number of the triple positive cells (profile) in dissociated ONPs transplantation vs. ONP spheroid transplantation with three different substrates. (d): Quantification of the number of neurites in dissociated ONPs transplantation vs. ONP spheroid transplantation. NIM: neuronal induction media, G: GrowDex®-T, G+P: GrowDex®-T and PODS®-hBDNF. *p < 0.05, ** p < 0.01, N.S.: not significant by one-way ANOVA with Tukey’s post-hoc test. Note that all of the images stained for TOTO3 iodide and AHNA have been pseudo-colored. (D): Calcofluor White staining indicates the presence of NFC at both seven days and ninety days post-transplantation. (E): Tissue response in H&E histology. (a) Control WT cochlea (no surgery). (b) DTR mouse cochlea transplanted with hESC-derived ONP spheroids with GrowDex®-T and PODS®-hBDNF. The DTR mouse was euthanized seven days after the transplant surgery (D10). (c) DTR mouse cochlea transplanted with hESC-derived ONP spheroids with GrowDex®-T and PODS®-hBDNF. The DTR mouse was euthanized 90 days after the transplant surgery (D90). (F): Expression of MHC class I and MHC class II proteins assessed by flow cytometry in undifferentiated hESCs (H9) and hESC-derived ONP spheroids (ONPs). Red lines represent background staining with the conjugated antibody (MHC I and MHC II) alone. Three independent experiments were performed and the results were averaged for each analysis.