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. 2021 Apr 13;30(8):399–417. doi: 10.1089/scd.2020.0085

FIG. 5.

FIG. 5.

Immunolocalization of 5mC and 5-hmC in early and late human retinal organoids and developing human fetal retina. (1) Similarity of 5hmC distribution in the 10 week-old hESC-derived retinal tissue from hESC line H1 and human fetal retina. In the hESC-derived retinal tissue (1.a1, 1.a4) and human fetal retina (1.b1, 1.b4) 5hmC staining (green) was localized to the periphery of the cell nuclei in the majority of cells in the INBL. The white square brackets in (1.a1, 1.b1) demarcate the INBL within the two tissues. However, in the ONBL only a few cells have 5hmC staining in the cell periphery (asterisk). Insets show magnification of the area marked with asterisks. In hESC-derived retinal tissue (1.a2, 1.a5) and fetal retina (1.b2, 1.b5) 5mC staining (red) was localized to the chromocenters of the cell nuclei. Nuclei are counterstained with DAPI. Insets in (1.b4–1.b6) (*) and (1.b3, 1.b4) (**) represent magnification of areas shown with asterisks (*), (**) in (1.b4, 1.b3), respectively. (1.a4–1.a6) Magnification of areas marked in (1.a1–1.a3) with asterisks. (1.a3, 1.a6, 1.b3, 1.b6) The merged image of 5hmC and 5mC staining. Scale bar: 20 μm. In hESC-derived retinal tissue (week 4), 5hmC and 5mC staining was mostly confined to the PR layer (1.c1–1.c6). 5hmC staining was present in the periphery of the cell nuclei (1.c1, 1.c4), whereas 5mC staining was localized in the chromocenters of the cell nuclei (1.c2, 1.c5). (1.c3, 1.c6) The merged image of 5hmC and 5mC staining. Nuclei are counterstained with DAPI. In human fetal retina (16 weeks), 5hmC staining was strongly localized in the INBL (1.d1, 1.d4) whereas 5mC staining was found in chromocenters in ONBL and INBL (1.d2, 1.d5). (1.d3, 1.d6) Merged image of 5hmC and 5mC staining. Nuclei are counterstained with DAPI. Insets represent magnification of the area shown with an asterisk in the images. (2) Percentage of 5mC in hESC-derived retinal tissue and human fetal retina. Error bar shows (±) standard deviation. ***P < 0.0001. 5hmC, 5-hydroxymethylated cytosine.