Table 1.
Antibodies used for staining target cells.
Fig 1.
Differentiation of human induced-pluripotent stem cells (iPSCs) into iPSC-derived retinal pigment epithelial (RPE) cells from 2 different donors.
Immunofluorescent staining was positive for Oct-4, Nanog, Sox-2, and TRA-1-60 (A). Nuclei stained with DAPI (blue). Undifferentiated human iPSC colony (B) and eventual formation into embryoid bodies (C). Formation of neural rosettes by day 14 post-differentiation (D), and a pigmented monolayer of iPSC-derived RPE cells forms by day 45 post-differentiation (E). Scale bar for A and E = 50 μm.
Fig 2.
Expression and localization of retinal pigment epithelial (RPE) cell markers in human iPSC-derived RPE.
Immunofluorescent staining was positive for bestrophin, Na + /K + ATPase, RPE65, and ZO-1 (A). Nuclei stained with DAPI (blue). Scale bar for A = 50 μm.
Fig 3.
Transepithelial resistance (TER) of iPSC-derived RPE cells on RPE cell-derived ECM and nitrite-modified RPE cell-derived ECM.
Monolayer permeability was assessed by TER in polarized iPSC-RPE cells. TER measurements were 209.5 and 209.3 Ω*cm2 for iPSC-derived RPE cells cultured on untreated extracellular matrix. iPSC-derived RPE cells cultured on nitrite-modified ECM were (225.8 and 207.3 Ω*cm2) from donor one and two samples, respectively.
Fig 4.
VEGF release in iPSC-derived RPE cell monolayers on RPE cell-derived ECM and nitrite-modified RPE cell-derived ECM.
iPSC-derived RPE cells release of vascular endothelial growth factor (VEGF) on nitrite-modified ECM (nitrite) and untreated ECM (PBS) both apically and basally. ****p <0.0001.
Fig 5.
PEDF release in iPSC-derived RPE cell monolayers on RPE cell-derived ECM and nitrite-modified RPE cell-derived ECM.
iPSC-derived RPE cell release of pigment epithelium-derived factor (PEDF) increased on nitrite-modified ECM (nitrite) apically. **** p <0.001.
Fig 6.
C3a production in iPSC-derived RPE cell monolayers on RPE cell-derived ECM and nitrite-modified RPE cell-derived ECM.
iPSC-derived RPE cell C3a production increased on nitrite-modified ECM (nitrite). * p <0.0001.
Fig 7.
Effects of nitrite modification on VEGF, PEDF and C3a in iPSC-derived RPE cells.
Release of VEGF, PEDF and c3a from patient-derived iPSC-RPE cells on normal versus “aged” extracellular matrix is represented diagrammatically. VEGF release is higher basally than apically on normal basement membrane (A). Cross-linking of collagen in the basement membrane increased release of VEGF both apically and basally in iPSC-derived RPE cells while preserving this polarity (A). PEDF release is higher apically than basally on normal basement membrane (B). Cross-linking of collagen in the basement membrane increased release of PEDF apically, and preserved this polarity (B). Cross-linking of collagen in the basement membrane increased release of C3a in iPSC-derived RPE cells (C). The aging of Bruch’s membrane increases VEGF (apical and basal) and PEDF (apical and basal) release in the RPE cells as well as C3a production. Increased release of these factors may contribute to the pathology at the site of RPE cells seen in exudative and advanced dry age-related macular degeneration (AMD) or geographic atrophy.