H1 (25) (National Institutes of Health–registered as WA01) were maintained in Matrigel (BD Biosciences, USA) using mTeSR (StemCell Technologies, USA) and subcultured using Dispase (BD Biosciences, USA). Differentiation protocol was performed by allowing H1 to overgrow until the hES colonies became multilayered. Culture media was then replaced with RPE differentiation medium (RPE medium), composed of knockout high glucose DMEM supplemented with 0.1 mg/ml Normocin (Invivogen, USA), 1% nonessential amino acids solution, 2 mM GlutaMAX-I (Invitrogen, USA), 0.1 mM mercaptoethanol (Invitrogen, USA), 13% Serum Replacement (Invitrogen, USA) and 5% Fetal Bovine Serum (FBS) (Cripion Biotecnologia LTDA, BRA) (Fig. 1A).

ARPE-19 cell line, previously described as a human RPE cell line, was cultured in DMEM/F12 supplemented with 10% Fetal Bovine Serum. The medium was changed every 2 days and cells were used between passages 9-19.

Primary RPE cells were isolated from fetal (18∼22 weeks gestation) and adult (59∼63 years old) eyes (Advanced Biosciences Resources, Inc, USA). Cells were obtained following RPE layer digestion using collagenase IV (Gibco, USA) at 0.8 mg/ml (fetal eyes) or 0.4 mg/ml (adult eyes). RPE cells were cultured using RPE medium and tissue culture flasks covered with the extracellular matrix produced by bovine corneal endothelial cells (26). After reaching confluence, cultures were expanded using 0.25% trypsin-EDTA.

Cells were harvested from fetal and adult RPE (fRPE and ARPE, respectively), ARPE-19 and hES-RPE cultures for mRNA characterization (Fig. 1B). fRPE were collected at Passage 1, day 4 for experiments. ARPE were dissected from a donor eye (age 59∼63) and directly processed for RNA isolation using Trizol. Total RNA samples were treated with DNase (Promega, USA) and quantified by spectrophotometry. cDNA was obtained using the RevertAid^TM H Minus M-MuLV RT (Fermentas, USA). Next, PCR amplification for pluripotent stem cell markers OCT-4 and NANOG, and RPE markers RPE-65, bestrophin, CRLBP, MITF, PEDF, and ZO-1, was performed using the TaqMan Gene Expression Assay kit (Applied Byosystems, USA). The relative level of gene expression was determined and normalized to 18s rRNA. Each sample was run in technical triplicates and biological dup-licates. Comparisons were made considering samples processed at the same time.

The protein expression of MITF and BEST was also evaluated using Western Blotting. Cell lysates were prepared from RPE-hESC in passages 1 to 3. The cells were lysed on ice using cold lysis buffer (20 mM Tris, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 10% glycerol) containing protease inhibitor (Protease Inhibitor cocktail II, Calbiochem). Then, samples were vortexed for 15 seconds and incubated on ice for 5 min. After repeating this step two more times, the lysate was centrifuged for 15 min at 13500 g at 4℃. Protein concentration was measured by Bradford assay (BioRad). Cell lysates (30 μg) were mixed with Laemmli buffer, added with β-mercaptoethanol and boiled for 5 min. Proteins were separated using 12% SDS-PAGE polyacrylamide gel and then transferred to a nitrocellulose membrane for 2 h at 75V in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3). The membrane was washed with PBS-T (Tween-20 0.05%) and blocked with 5% BSA in PBS-T for 1 h. The membranes were incubated with mouse anti-RPE-65 1：500 (Pierce) and mouse anti-CRLBP 1：750 (Abcam) overnight at 4℃. After 3 washes of 15 min in PBS-T, the membranes were incubated with secondary antibody anti-mouse IgG diluted 1：10,000. Chemiluminescence was developed using the ECL Plus kit (Amersham Biosciences) and the signal scanned and collected using a Typhoon imager (Amersham Biosciences).

RPE markers were also analyzed in hESC-RPE using immunofluorescence. For this, passage 2 RPE cells were fixed with 4% paraformaldehyde for 15 min, permea-bilized with 0.1% Triton X-100, and blocked with a solution containing 2% goat serum (Normal Goat Serum - NGS) 0.5% BSA, and 0.1% Triton X-100 diluted in PBS for 1 hour. Primary antibodies were diluted in 2% NGS / PBS-T 0.3% and incubated at 4℃ overnight, using the following dilutions: 1：50 of the rabbit polyclonal transcription factor associated with microphthalmia, MITF (Abcam); 1：250 of mouse monoclonal bestrofin membrane protein, BEST (Pierce). Goat anti-mouse IgG FITC (1：32; Sigma), and goat anti-rabbit IgG rhodamine (Jackson ImmunoResearch Laboratories) were diluted in blocking solution and incubated with samples for 1 hour at room temperature. Nuclear staining was executed using 0.2 μg/ml DAPI (Sigma) diluted in PBS. The slides were assembled using Vectashield (Vector Laboratories). The images were obtained using a confocal microscope (Confocal Zeiss 5 LIVE).

Melanin content is one of the criteria used to select and characterize hESC-RPE batches used for cellular therapy (27-29). In order to quantify the intracellular melanin content of hESC-RPE, passage 2 cells were harvested on days 8, 12 and 16 after passage. The cells were centrifuged at 160 g for 5 min at room temperature and counted. Pellets were resuspended in 1M NaOH and heated at 80℃ for 10 min, vortexed, and the absorbance measured at 475 nm against a standard synthetic melanin curve (Sigma) ranging from 5 to 180 μg/ml. Samples were analyzed in triplicates and the data normalized to the total number of cells.

hESC-RPE were plated at 6×10⁴ cells/cm² on Transwell plates (Corning) prepared with Matrigel or BM substrates (Sigma). The cell culture supernatants on the apical and basal sides (the compartment above and below the Transwell, respectively), were collected 48 hours after plating and the samples assayed, in duplicates, with the DuoSet ELISA Human VEGF R&D Systems kit.

The ability of ARPE-19 and hES-RPE cells to resurface aged human BM was assessed since this is the area where RPE is first affected in diseases like AMD, and also as an additional evidence of the resemblance of ARPE-19 and hES-RPE to the native RPE. In this assay, cellular attachment and survival were analyzed by scanning electron microscopy (SEM).

In order to prepare the ex vivo culture experiment, adult donor eyes (age 59∼63) were received from the eye bank of the city of Belo Horizonte, MG - Brazil (Ethical Committee approval no. ETIC 33734514.7.0000.5149). Acceptance criteria followed previous studies (30, 31). Six-millimeter-diameter corneal trephines (Bausch and Lomb, USA) was used to create macula-centered BM explants which were debrided. Explants were seeded with 3,164 cells/mm², shown to yield a monolayer of cells with 24 h after seeding (30). Explants were harvested at day 7, fixed in paraformaldehyde-glutaraldehyde Karnovsky’s Fixative solution and processed for SEM.

Scanning electron microscopy: Explants were fixed as mentioned and washed 3 times with PBS and incubated with 1% osmium tetroxide (Electron Microscopy Sciences, USA) in PBS at 4℃ in the dark for 1 h. After, samples were washed with PBS and incubated in 1% tannic acid in water. Explants were subsequently washed with PSB and incubated in 1% osmium tetroxide in water at 4℃ in the dark for 1 h. Then, samples were washed with PBS and dehydrated through stepwise incubation in a series of graded ethanol baths at 35%, 50%, 70%, 85%, 95% and 100% concentration. Samples were submitted to critical point drying using CO₂ (Leica EM CPD030, USA). Finally, explants were coated with gold (BALTEC MED020 Coating System, LIE) and analyzed in the Microscopy Center of Biological Sciences Institute, Federal University of Minas Gerais, Brazil (DSM950- Zeiss, GER).

hES-RPE cells and ARPE-19 were seeded on 96-well plates at 1×10⁴ cells/well. After 24 h, the medium was changed and CSA, SRL, TAC, LEF, and TER were added at increasing doses of 31.6 μM (10^1.5), 56 μM (10^1.75), 100 μM (10²), 177.8 μM (10^2.25) and 316 μM (10^2.5). Cells were incubated with respective drugs for 72h and had their viability assessed by the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) assay (32) or CellTiter Blue, following manufacturer’s instructions. Data were obtained from three independent experiments. The IC50 (concentration of the drug in which cell viability decreased by 50%) was determined for each drug incubated with each cell.

Statistical analysis was performed by analysis of variance (ANOVA) followed by Bonferroni’s post-test using Graph Pad Prism 5.0. Values were represented as mean – standard deviation. Differences were considered significant if p<0.05.

hES-RPE and ARPE-19 are considered relevant experimental models of the native RPE, for in vitro investigations. In order to assess the similarity of hES- RPE and ARPE-19 relative to fRPE and in situ ARPE, these cells were obtained and characterized morphologically, molecularly and functionally (Fig. 1B, Supplementary Fig. S1). mRNA expression of proteins involved in visual cycle (CRLBP and RPE-65), RPE-specific transcription factors (MITF), membrane-associated proteins (BEST), secreted factors (PEDF) and tight junction proteins (ZO-1) was performed. Obtained data showed that hES-RPE mRNA levels were similar to fRPE and ARPE (Fig. 2A). On the other hand, ARPE-19 revealed lower expression levels of all RPE markers compared to other groups, with ZO-1 being the only exception, presenting statistically similar expression between ARPE and ARPE-19. Despite constituting an important gene for RPE function, due to its contribution to tight junction formation and integrity of the blood-retina barrier, ZO-1 is not a specific RPE marker. Compared to undifferentiated pluripotent stem cells, hESC-RPE presented lower expression of pluripotency markers, as well as higher expression of RPE markers (Fig. 2B).

The RPE differentiation was also investigated using protein markers. Immunofluorescence analysis confirmed that hESC-RPE cells expressed BEST, MITF, with membrane and nuclear localization, respectively (Fig. 2C).

Finally, the hESC-RPE differentiation was confirmed using western blotting analysis of RPE-65 and CRLBP, which were expressed by hESC-RPE in different passages, similar to fRPE cells (Fig. 2D).

Pigment production is also a determining factor in the maturation stage of cells, this being one of the criteria used to determine the degree of differentiation that cells should be used for therapy. Therefore, the functionality of hESC-RPE was analyzed according to the melanin content of cells in different time-points of differentiation (Fig. 3A), and revealed that the differentiated cells accumulated intracellular melanin over time. The verified melanin content surpassed the minimal hESC-RPE melanin content for clinical application (27-29).

RPE cells in situ are known to secrete growth factors, such as VEGF, in a polarized manner (10). Similarly, RPE monolayers cultured in vitro show preferential secretion of VEGF in the basal side (33). In order to verify the functional character of the EPR-hESC cells, they were seeded in transwells and the culture medium was collected from both upper and lower reservoirs, as previously described (34). The hESC-RPE secreted VEGF preferentially in the basal side, with a basal/apical ratio ranging from 7.4 to 9.9 (Fig. 3B). The production of VEGF on both the apical and basal side was similar for cells cultured on both types of MEC substrates of Matrigel and also BM. The resurfacing experiments of hBM are further described below.

The capacity of hES-RPE and ARPE-19 to resurface the BM surface was assessed in order to investigate their ability to attach and survive on the area where RPE cells are critical for the maintenance of vision, the macula. To do so, hES-RPE and ARPE-19 were seeded on paired donor aged BM after a debridement protocol (Fig. 4A and 4B). SEM analysis revealed that ARPE-19 cells failed to attach and survive in this biologically relevant substrate, once only a few cells could be seen spread on BM (Fig. 4C and 4D). On the other hand, not only hES-RPE cells were able to completely resurface BM but also revealed a morphological resemblance to the native RPE (Fig. 4E and 4F).

Following the introduction of in vitro assays, which aroused as alternative methods for in vivo toxicity assessment, several groups moved to in vitro models to test drug toxicity and efficiency (4). Since our previous experiments have highlighted phenotypic and functional differences between hES-RPE and ARPE-19, we proceeded with the analysis of another possible difference between those cells - their sensibility to specific drugs. Our data showed that CSA, SRL, TAC, LEF, and TER significantly decreased cell viability in vitro in a dose-dependent manner (Fig. 5 I e II). Interestingly, for most of the tested drugs, ARPE- 19 cells viability was accentuated reduced compared to hES-RPE cells, particularly at higher drug dosages. Indeed, according to MTT cell viability assay, TER treatment (Fig. 5 IE and IIE) was significantly toxic to ARPE- 19 at >31.6 μM (p<0.0001), while to hES-RPE cells, significantly decreased cell viability at >100 μM (p< 0.0001) (IC₅₀ values were 133.2 μg.ml⁻¹ for hES-RPE and 103.9 μg.ml⁻¹ for ARPE-19). LEF treatment (Fig. 5 ID and IID) results also confirmed this evidence and showed that, considering each tested concentration of this drug, ARPE-19 showed less cell viability level than hES-RPE cells, for MTT and CellTiter-Blue^Ⓡ cell viability assays. LEF significantly reduced cell viability in both assays and for both cells at concentrations >31.6 μM (p<0.0001), and showed IC₅₀>316 μM for hES-RPE cells, while for ARPE-19 the IC₅₀ value was 166.70 μM.

Regarding SRL (Fig. 5 IB and IIB), the most toxic drug among those evaluated, we observed different cytotoxic effects on retinal cells at the lowest concentration of this chemical, according to the MTT cell viability assay. However, at 56 μM, hES-RPE cells were more resistant to SRL compared to ARPE-19 cells, for both assays. SRL was significantly toxic for ARPE-19 cells at concentrations >56 μM (p<0.0001), and >100 μM for hES-RPE (p< 0.0001) (IC₅₀ value of 75.1 μg.ml⁻¹ for hES-RPE while the IC₅₀ value for ARPE-19 was lower, 48.7 μg.ml⁻¹).

On the other hand, the results suggested that hES-RPE cells were more sensitive to CSA and TAC treatments, considering resorufin assay. TAC treatment (Fig. 5 IC and IIC) significantly reduced cell viability on both cell lines at concentrations >100 μM (p<0.0001), showing IC₅₀= 127.74 μM for ARPE-19 and IC₅₀=117.03 μM for hES- RPE. The CSA treatment (Fig. 5 IA and IIA) was the least toxic drug tested and the viability of the tested cells was statistically different at concentrations >10 μM (p= 0.0093) on CellTiter-Blue^Ⓡ assay, showing IC₅₀>316 μM for ARPE-19 and 260.4 μM for hES-RPE. Even with these differences, MTT assay demonstrated at higher concentrations of these drugs that ARPE-19 cells were more sensitive to all drugs tested. Therefore, it can be observed that, overall, ARPE-19 revealed higher sensibility to toxicological assault than hES-RPE.