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. Author manuscript; available in PMC: 2024 Jul 6.
Published in final edited form as: Cell Stem Cell. 2023 Jul 6;30(7):950–961.e7. doi: 10.1016/j.stem.2023.06.006

Figure 1. PAX2-2A-nGFP/POU4F3-2A-ntTomato (PAX2nG/POU4F3nT) multiplex reporter hESCs faithfully recapitulate otic progenitor and hair cell differentiations in inner ear organoids.

Figure 1.

(A) Schematic illustrations of the PAX2-2A-nGFP and POU4F3-2A-ntdTomato CRISPR design.

(B) Schematic of PAX2nG and POU4F3nT reporter expression during inner ear organoid development.

(C-F) Live images of whole aggregates containing multiple developing inner ear organoids show the spatio-temporal progression of PAX2nG reporter expression and early morphogenesis of PAX2+ epithelium.

(G-H) Representative images of hESC-derived aggregates showing PAX2nG+ epithelium organized into vesicles that co-express the otic-specific marker FBXO2, but devoid of POU4F3nT expression.

(I-I’) Live images of late-stage (D96) aggregates showing intense POU4F3nT+ puncta localized to epithelial vesicles.

(J-K) POU4F3nT+ cells in inner ear organoids also express the hair cell markers MYO7A and SOX2, and are located on the luminal surface of SOX2+ supporting epithelia.

(L) Fixed cell suspension of dissociated POU4F3nT+ cells isolated from D80 inner ear organoids reveals tdTomato+ nuclei and F-actin+ membranes and stereocilia.

Scale bars, 200 μm (C-G, H, I, I’), 50 μm (G’, H’), 10 μm (J-L).