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. 2021 Oct 26;15:752958. doi: 10.3389/fnins.2021.752958

FIGURE 1.

FIGURE 1

Generation of hESC derived retina organoids (CRX-GFP cell line) and RPE (H9 cell line). (A) Phase contrast images of organoid development up to day 56. (B–D) Progenitor gene expression analysis of CRX-GFP hESC-derived retina organoids (n = 2) and undifferentiated stem cells (n = 1). (B) OCT4 expression is absent in differentiated day 48 retina organoids. (C) Retina organoids (red) highly express genes indicative of primitive retina and photoreceptor progenitors. (D) Markers for mature, terminally differentiated retinal subpopulations. Retina organoids (red) contain markers for all the major retinal cell types, including photoreceptor progenitors, glia, ganglion cells, bipolar, horizontal, amacrine, and Müller cells. (E) Flow analysis shows that GFP expression is absent in the undifferentiated stem cells, early embryoid bodies, and young retina organoids. A distinct GFP+ population appears at day 70. Expression is highest day 78 and persists in the oldest analyzed day 91 organoids. (F) Examples of gates drawn used for FACS sorting of the GFP negative and GFP positive populations. (G) Post-FACS purities >90%. Cells were gated based on singlets, SSC vs. FSC profile, and if they were DAPI negative. Negative controls were undifferentiated CRX-GFP hESC (day 0). (H) Parylene only implant (no cells). (I) CPCB-RPE1 implant covered with hESC-RPE cells. (J) Enlargement of (I) to show details of hESC-RPE cells forming an epithelium (28–35 days culture).