Figures and data
iPSC RPE morphology and marker expression is altered by media composition.
(A) Formulation of Media 1 – 6 used in this study. (B) Representative brightfield images of iPSC RPE cultured for up to 8 weeks on Matrigel coated culture dishes. (C,D) iPSC RPE cultured on chambers slides were fixed and immunostained for ZO-1, BEST1, and PMEL. (E) RPE cell area and hexagonality were calculated using REShAPE analysis. (F,G) Immunoblotting and quantification of key RPE markers in RPE lysate after 8 weeks culture in Media 1 – 6. Mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001.
Nutrient composition influences RPE polarity and sub-RPE deposition.
(A) TER of iPSC RPE over time on 0.33cm2 PET Transwell filters, and (A’) at 8 weeks. (B-D) ELISA of VEGF, ApoE and CFH in 24h conditional media from RPE cultured on flat bottom dishes. Mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001. (E) Live cell stain with Lipi-DeepRed (Lipi-DR) and BODIPY-493/503 to identify lipid droplets in the 8 weeks iPSC RPE. Scale bar, 10μm. (F) Representative TEM images of apical microvilli and basal infoldings with sub-RPE deposits in 8 weeks iPSC RPE on Transwell filters. *, amorphous deposits; ^, banded collagen. Scale bar, 500nm. (G) Quantification of lipid droplets per 100μm2 area in Lipi-DR and BODIPY images. (H) Sub-RPE deposits were identified and quantified in n=3 panoramic sections of the TEM images.
Respiration, glycolysis and extracellular metabolite consumption/production in iPSC RPE.
(A,B) iPSC RPE were seeded and maintained on Matrigel coated XFe96 well plates in respective Media 1 – 6 for four weeks. Prior to (A) mitochondrial stress test and (B) glycolysis stress test, cells were equilibrated in XF DMEM media (pH 7.4) in CO2-null incubator for 1 hour. Mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001. (C-K) Targeted metabolomics on 8-week-old iPSC RPE was performed via LCMS. (C) Extracellular metabolites in unspent media were plotted as a heat map, with degrees of grey depicting lowest to highest intensity. (D) To calculate metabolite changes in 48h spent media from iPSC RPE, results are depicted on a sliding scale where % metabolite = (Spent media – Unspent media)/Unspent media x 100. Green, production; Red, consumption; +, new production; ab, absent. Venn diagram showing common and unique metabolites (E) consumed or (F) produced across all media between iPSC RPE and fRPE. (G) Newly produced metabolites that were initially absent in unspent media normalized to Medium 3. (H,I) Consumption rate and production rate of select metabolites plotted against abundance in unspent media. Media number noted in graph in grey. (J,K) Comparison of Medium 1 vs 2 and Medium 2 vs 3, to show changes in usage of metabolites when FBS is replaced with B27.
Heatmap of intracellular metabolites involved in lipid, nucleotide, NAD, vitamin metabolism and TCA cycle in iPSC RPE and fRPE.
(A) 114 detected intracellular metabolites were categorized by major metabolic pathways, sorted left to right from highest to lowest abundance, normalized to Medium 1 and depicted as a heatmap. Yellow, fold change (FC)=1; Red, FC<1; Green, FC>1. Ratio of NAD/NADH and ATP/ADP in lysate of (B,C) iPSC RPE and (D,E) fRPE maintain in Media 1 – 6 at 8 weeks. Mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001.
Intracellular metabolites of amino acid and sugar metabolism.
(A) Heatmap of metabolites are analyzed as described in Figure 5. (B) Ratio of GSH/GSSG in iPSC RPE and fRPE. (C) Histogram of amino acid abundance normalized to medium 1. (D) Select intracellular metabolites were plotted against extracellular abundance in 48h spent media. All data normalized to medium 1, except for 2-methyl-butyroylcarnitine, which was normalized to medium 3. Mean ± SEM. *, p<0.05; **, p<0.01.
