Figure 6.

Genetic Correction Restored Hyperpolarization-Activated Potassium Current (I_h) in Cells from Patient Retinal Organoids at W36

(A–C) Representative traces of current responses elicited by a series of voltage steps from −110 mV to 30 mV with a 20 mV increment (protocol shown as inset). Notice I_h (arrow) in cells recorded from control retinal organoids (A), which was absent from patient ones (B) and restored in corrected ones (C).

(D) BF and fluorescent (filled with Alexa 555) images of a recorded cell located at the outer layer of retinal organoids.

(E) Comparison of I-V curves of cells recorded from control 1 (blue, n = 24), patient 1 (orange-red, n = 21), and corrected 1 retinal organoids (green, n = 15). I_h, activated between −80 mV and −110 mV, was significantly larger in cells from control and corrected organoids, and markedly reduced by the application of 40 M HCN blocker ZD7288 (wild-type [WT] + ZD7288, gray). Asterisk (^∗), patient retinal organoids significantly different from control and corrected. Hash (#), significant difference between WT and WT + ZD7288. Results are pooled from three independent experiments. Data are presented as mean ± SEM. Statistical significance was determined using unpaired t test.

(F) Heatmaps illustrate the gene expression profile of HCN-1 in RNA-seq dataset at week 22. Different colors represent the value of log2 (FPKM+1). The size of dot represents the FPKM ratio of corrected to patient, control to patient, and corrected to control. Orange dots, >2 fold changes; green dots, approximately 1.5–2 fold change; blue dots, <1.5 fold change.

(G) Sample images of the immunostaining of patient, corrected, and control retinal organoids at W22. Scale bar, 7.5 μm.