RGC axonal outgrowth and pathfinding into assembloids
(A) Retinal organoids expressing POU4F2:tdTomato were fused to CTIP2-positive brain organoids at 50 days of differentiation to analyze the extent of RGC axonal outgrowth into assembloids.
(B) Schematic of the method for quantifying axonal outgrowth from 3 days post-assembly (dpa) to 7 dpa.
(C) By 3 dpa, axons had begun to extend into brain organoids.
(D) More extensive RGC axonal outgrowth was observed by 7 dpa.
(E) By 5 dpa, significantly more axons had reached the midpoint of brain organoids or beyond, with a majority of axons reaching the end of brain organoids by 7 dpa.
(F) RGC axons displayed pathfinding abilities, with more axons (arrows) avoiding SOX2-positive ventricular-like zones (dotted lines), rather than extending through.
Error bars represent SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗p < 0.001. n = 6 separate differentiation experiments each using the H7 and H9 lines of hESCs. Scale bars, 250 μm.