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. 2021 Mar 18;15:648210. doi: 10.3389/fncel.2021.648210

Figure 1.

Figure 1

Optogenetic engineering of retinal structures derived from human induced pluripotent stem cells (hiPSCs). (A) Bright-field/epifluorescence image of a hiPSC-derived retinal organoid after mechanical isolation on day 30 of differentiation. (B) Schematics of the adeno-associated viruses (AAV)-mediated strategy used to engineer 3D retinal organoids, AAV2–7m8-PR1.7-Jaws-GFP was used to transduce cone photoreceptors on day 44 of differentiation. (C) Live GFP fluorescence observed after prolong culture of retinal organoids showing strong and long-term expression of the transgene. (D) Merge of bright-field/epifluorescence and live GFP fluorescence images observed in 70-days old organoids. (E) Immunofluorescence (IF) analysis of a representative retinal organoid at day 70 of differentiation, depicting a thick layer of photoreceptors immunoreactive for GFP and some sparse ganglion precursors cells identified by BRN3a. (F) Magnification of the IF analysis in (D) in another organoid. (G) Real-time qRT-PCR analysis of cone specific marker, Cone arrestin (ARR3) and retinal ganglion cell specific marker BRN3B. N = number of biological replicates. Values are mean ± SEM. Error bars are SEM. Statistical significance assessed using Mann–Whitney Student’s test (***p < 0.0005). (H) IF analysis of optogenetically engineered retinal organoids at day 70 showing Jaws+ cells do not express CD73 photoreceptor surface marker. (I) IF analysis confirming postmitotic photoreceptor identity of GFP positive cells, showing they are immunoreactive for the photoreceptor-specific marker Recoverin (RCVN) and complete absence of proliferation marker KI67. (J) Magnification of the IF analysis in (I) in another organoid. (K) IF analysis of confirming the cone identity of GFP positive cells, showing colocalization with cone-specific marker GNAT2. (L) 2-Photon laser image of GFP+ cells inside a retinal organoid at D70 of differentiation. Images correspond to representative retinal organoids from at least four different biological replicates, scale bar: (C,D) = 250 μm, (E,F,I) = 100 μm, (H,J,K) = 50 μm, (L) = 10 μm.