Fig. 4: The controlled release of Wnt-surrogate in an organoid internal gradient permits the bioengineering of RO with a more in vivo-like retinal cell type diversity.

a, Representative confocal transmission and maximum intensity z-projection (Atoh7::EGFP; 35 slices at 5 µm distance) images of day 4 RO treated with 0 nM, 1 nM and 4 nM Wnt-surrogate in the culture medium. b, Quantification of the area of RPE (black) and retinal ganglion cell numbers (green) from representative transmission and Atoh7::EGFP maximum intensity z-projection images obtained as in a. Each box plot contains data from n = 5 RO. c, Representative confocal transmission and maximum intensity z-projection (Atoh7::EGFP; 15 slices at 10 µm distance) images of day 4 RO after Wnt-DNA microbead microinjection and Wnt-surrogate release at day 1. White dashed lines outline the shape of the respective RO. d, Quantification of the area of RPE (black) and retinal ganglion cell numbers (green) from representative transmission and Atoh7::EGFP maximum intensity z-projection images obtained as in c. Each box plot contains data from n = 7 RO for Wnt-DNA microbeads −UV and n = 10 RO for Wnt-DNA microbeads +UV. Boxes indicate 25–75% percentiles and whiskers 10–90% percentiles. The central horizontal line indicates the median. Individual data points shown as dots. Retinal ganglion cell numbers were normalized to the average of all data points of the 0 nM or Wnt-DNA microbeads −UV group, while the area of RPE was normalized to the average of all data points of the 4 nM or Wnt-DNA microbeads +UV group. Two-tailed Student’s t-tests were performed with unequal variance (NS, not significant; *P value 0.03; 0 nM versus 1 nM ***P value 0.0003; 1 nM versus 4 nM ***P value 0.0004; Wnt-DNA microbeads +/−UV ***P values 0.0002). e, Live epifluorescence microscopy of a day 4 RO after Wnt-DNA microbead microinjection and Wnt-surrogate release at day 1 near the RO’s edge. Note that the RPE induction phenotype was deliberately reduced by changes in culture conditions to more precisely show the spatial relationship of the DNA microbeads and RPE differentiation. Magenta dashed lines indicate RPE. Scale bars, 100 µm.