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. 2016 Apr 28;6(5):743–756. doi: 10.1016/j.stemcr.2016.03.011

Figure 4.

Figure 4

Self-formation of Retinal Organoids Comprising Apically Convex VSX2+ Epithelium and PAX6high RPE Progenitors in Floating Cultures

(A) Live view of retinal organoids at D13 + 6D. Note the translucent areas (arrowheads). 400–600 spheres per 6-well plate of H1 hESCs, n > 10 independent experiments.

(B) A high-magnification view of a D13 + 9D retinal organoid with a bright ring (arrow in B).

(C) RT-qPCR with RNA isolated from three individual wells of adherent cultures on D13, four individual wells of adherent cultures on D13 + 13D, and four groups of retinal organoids on D13 + 13D (15 pooled retinal organoids in each group). The expression was represented as fold changes relative to that in adherent cultures on D13. Mean ± SD of four or three individual wells of adherent cultures or groups of retinal organoids. Expression changes between D13 and D13 + 13D adherent cultures indicated cell differentiation in time course. Expression pattern in retinal organoids consistently differed from that in adherent cultures on D13 + 13D: the expression of VSX2, SIX6, and TJP1 was higher, but the expression of OTX2 and SNAI2 was lower. Ad., adherent cultures; ROs, retinal organoids.

(D–I) Immunostaining of the retinal organoid at D13 + 9D (B). VSX2 was highly expressed in the epithelium (D, 90%). VSX2+ cells expressed SIX3 and PAX6, but not OTX2 (arrows in E–G). The peripheral cells with low or no VSX2 highly expressed PAX6 and OTX2 (arrowheads in F and G). TJP1 and PRKCZ were at the convex surface of VSX2+ epithelium (arrows in H and I). LAMB1 expression was variable (arrowhead in I). A stalk connected the outer and inner epithelium (diamond arrow in I).

(J–O) VSX2+ RPCs sorted out from OTX2+ cells and self-organized into an apically convex epithelium in aggregates at D14 + 14D. VSX2 was specifically expressed in the epithelium with TJP1 at convex surface, whereas OTX2 was expressed in VSX2− regions with TJP1 at concave surface (lumens) (arrows and diamond arrows in J, K, and M). LAMB1 was found at the basal surface of internal VSX2+ epithelium (arrowhead in K) and amorphous areas (diamond arrowhead in K). TUBB3 was expressed (diamond arrows in N). VSX2+ RPCs co-expressed PAX6 and SIX3 (arrows in J, L, and O). Scale bars, 500 μm (A), 100 μm (B, D–I) and 200 μm (J–O). See also Figure S4 and Movie S2.