Fig. 7

Functional characterization of the in vitro-derived hair cell-like cells. a Organoid derived from a W9.6 sample (E1286), stained for ESPIN and BRN3C after FM1–43 labeling. Scale bar 10 μm. The same cell marked by the asterisk is shown in the inserts. b Hair cell-like cell stained for ESPIN and BRN3C after FM1–43 labeling. Scale bar 10 μm. Gain values for FM1–43 are set in order to visualize intracellular dye loading. c Re-image with lower gain of ESPIN+ bundle and FM1–43. d Hair cell -like cells derived from a EPCAM+/CD271+ organoid from a W12 sample (E1289) stained for MYO7A and F-actin after FM1–43 loading. Cell volumes defined in the MYO7A channel (dashed lines) were used to assess fluorescence intensity in the FM1–43 channel as shown in Supplementary Fig. 9. Scale bar 10 μm. For all examples (a–d) FM1–43 uptake was performed with a 30 s exposure in presence of concanavalin A. e Hair cell-like cells in an organoid derived from an EPCAM+/CD271+ sample (W9.6, E1286) showing GTTR uptake. The sample was incubated for 30 min with GTTR, followed by fixation and immunostaining for MYO7A and staining with phalloidin to visualize bundles. 3D projection of a confocal stack is shown for the single channel and merged colors. Boxed area indicates developing MYO7A positive cells. Scale bar = 100 μm. f 3D reconstruction of the area marked in e by the asterisks. Merged image f and red channel g are shown. Scale bar 10 μm