Fig 2: Immunofluorescent staining of RPE differentiation markers, PMEL-17 (C, K; green) and RPE65 (G, O; magenta) and BM markers, type IV collagen (B, J; red) and laminin a5 (F, N; yellow) in cryopreserved ihAM (A–H) and hESC-RPE cells culture on dhAM following thermolysin treatment (I–P). RPE markers identified differentiated hESC-RPE cells over dhAM (K, O), while BM markers demonstrated the preservation of the BM following thermolysin treatment (J, N). Merged images showed areas of membrane where RPE markers were not detected, despite a proper distribution of the BM markers (L, P). ihAM was used as control. hAECs, RPE cells and stromal cells nuclei were stained with DAPI (blue). Scale bars=100 µm. BM, basement membrane; dhAM, denuded hAM; hAM, human amniotic membrane; hESC, human embryonic stem cell; ihAM, intact hAM; RPE, retinal pigment epithelium.

Fig 3: Immune retinal infiltrates found in perivascular (a–j) and subretinal (k–t) areas of animals from group 3 and group 1, respectively. H&E staining shows the appearance of leukocytic cells infiltration in the perivascular area of a retinal blood vessel a (black arrow) and between the neural retina and the choroid k (black arrow). RPE65 staining shows the state of the RPE layer f and p being completely absent in the area where the subretinal infiltrate is present p. CD20+ and CD3+ cells b to e and l to o together with microglia activation i and s were found in both types of infiltrates. The macrophage marker CD68 was only detected in the perivascular infiltrate h. Scale bar: 30 µm. H&E, hematoxylin and eosin; ONL, outer nuclear layer; PR, photoreceptors; RPE, retinal pigment epithelium.

Fig 4: Immunohistochemical analysis of sham- (a–l) and rAAV8-treated (m–x) eyes from group 3. The 60 degrees fundus IR image showing the representative HRF distribution maps (a, m) with overlying position of B-scan (green line) and cross-sectional SD-OCT B-scans (b, n) demonstrate the appearance of HRF within the treated area 90 days after subretinal injection. Normal H&E staining of an area close to the SD-OCT scan (c, o). Immunohistochemical permanent staining of RPE65 shows the integrity of the RPE layer (d, p). B- and T-cells (CD20+ and CD3+ cells, respectively) were not detected in the sham treated eyes (e–h) but they were found in the rAAV8 treated eyes (q–t). The macrophage marker CD68 was not detected neither in the sham- (j) nor in rAAV8-treated (v) eyes. However, microglia cells were more active in rAAV8-treated eyes (w) than in sham-treated eyes (k). Scale bar: 50 µm. HRF, hyper-reflective foci; H&E, hematoxylin and eosin; IR, infrared; ONL, outer nuclear layer; PR, photoreceptors; RPE, retinal pigment epithelium; SD-OCT, spectral-domain optical coherence tomography.

Fig 5: Representative HE and immunostaining images of immunodeficient RCS rat retinas implanted with iPSC-RPE monolayer assessed at 11 months post-implantation. Presence of fibrosis, immunoreactivity, and epithelial–mesenchymal transition (EMT) was assessed. (a) Retina with no surviving iPSC-RPE showing signs of inflammation and peri-membrane fibrosis indicated by white asterisk. (b) Absence of TRA-I-85 staining. (c) Retinas showing RPE65 expressing iPSC-RPE cells (white arrows) labelled for GFAP (glial cells), (d) CD68 (macrophages/microglia), (e) expression of classical mesenchymal markers α smooth muscle actin α SMA and vimentin. (f) Images in which iPSC-RPE monolayer appears to be present below the parylene membrane are either due to orientation difference in the implant placement or due to the survival of the RPE on the lower side of the parylene membrane.