Figure 6: Evidence of Type I hair cells in ES cell-derived organoids.

(a) Confocal image of a day 28 organoid reveals numerous Myo7a+, Calb2- hair cells (arrowheads). (b,c) High magnification image of a Myo7a+, Calb2- Type I-like hair cell in a (asterisk). (d–f) DIC images of organoid hair bundles with Type II- (d,e) and Type I- (f) like morphology. Type II- (e) and Type I- (f) like voltage-dependent currents were recorded from the cells at the centre of these images (arrows). (g) Family of low-voltage-activated K⁺ currents, typical of vestibular Type I hair cells. The current is active at rest, deactivates with hyperpolarization (arrow) and activates further with depolarization. (h) Instantaneous tail currents at −35 mV plotted as a function of prepulse voltage for three Type I-like organoid hair cells. The data were fit with Boltzmann functions where V_1/2=−90, −61 and −86 mV. (i) Membrane responses of a utricle Type I hair cell and a Type I-like organoid hair cell evoked by the current steps shown below. (j) Family of membrane responses from a Type I-like organoid hair cell evoked by the current injection protocol shown (compared with Fig. 4m). (k) Summary time line of the functional maturation of mechanotransduction and ion channel acquisition for utricle hair cells (blue) and organoid hair cells over time in culture (red). Scale bars: 25 μm (a), 10 μm (d,e and f) and 5 μm (b,c).