Figure 3. PUR+IWP2 treatment promotes ventralization of otic progenitors in human inner ear organoids.

(A) Schematic illustration of known ventralization and dorsalization signals during mouse inner ear development and application of this principle to the human inner ear organoid system. PKA: protein kinase A, W: human gestational week, E: mouse embryonic day.

(B-D) D20 scRNA-seq analysis of FACS-sorted PAX2^nG+ otic progenitors in human inner ear organoids. UMAP projections of otic progenitors from PUR+IWP2, PUR and CTRL samples (B). Feature plots demonstrate that dorsal otic markers are predominantly expressed in PUR and CTRL otic progenitors, while ventral otic markers and SHH signaling components are confined largely to PUR+IWP2 cells. Consistent with this, the volcano plot (C) shows differentially expressed dorsal and ventral otic marker genes between PUR+IWP2 and CTRL otic progenitors. Gene-set enrichment analysis of genes upregulated in the PUR+IWP2 and CTRL otic progenitors (above and below 0 in the bubble plot, respectively) (D) shows genes associated with posttranscriptional regulation of gene expression, chromatin modifications and Hedge Hog signaling are enriched in ventralized otic progenitors in inner ear organoids.

(E) Representative images of D25 samples show notably higher expression of NR2F1 and SUlF1 in PUR+IWP2 organoids vs. CTRL organoids.

(F) Quantitative analysis of vesicles co-expressing PAX2 and NR2F1 (or SULF1) in PUR+IWP2 vs. CTRL organoids; n = 8 biological samples from separate experiments per group; Welch’s two-sided t-test *P=0.0013 (NR2F1), *P=0.0089 (SULF1); values are mean ± SEM.

Scale bars, 200 μm.