
Ocular toxicity tests are required to evaluate risks and ensure the safety of ophthalmic administration of drugs (1, 2). Several
The retinal pigmented epithelium (RPE) is a monolayer of pigmented epithelial cells that reside between the neural retina and Bruch’s membrane (BM). Even though RPE is not an intrinsic component of the visual signaling pathway, it is a highly metabolically active cell layer, which is vital to the health, survival, and function of the overlying photoreceptors (10, 11). Considering that RPE is critically important for normal function of the retina, intraocular drug or compound administration must be evaluated regarding possible toxicity against this cell layer (1, 12).
ARPE-19 was established and characterized in 1996 (6). Despite being considered a representative RPE cell line, these cells display poor transepithelial resistance values of ∼100 Ω.m2 and seem to lose RPE-specific genes when maintained in suboptimal culturing conditions (13). These limitations have encouraged the search for protocols for
Several diseases that cause ocular inflammation, including uveitis, scleritis, and orbital inflammatory disease result in impairment or loss of vision (17). The mainstay treatment is the use of corticosteroids, but the prolonged treatments and high doses of these drugs are associated with significant side effects (18). For this reason, corticosteroid-sparing agents like Cyclosporin (CSA) (19), Sirolimus (SRL) (20), Tacrolimus (TAC) (21), Leflunomide (LEF) (22) and its active metabolite teriflunomide (TER) have been investigated as alternatives to the use of corticosteroids.
While CSA, SRL, and TAC have already been applied for ocular diseases, there are few studies investigating LEF for this purpose (22-24). Nevertheless, the effect of these chemicals on RPE has not been verified
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 RevertAidTM 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.
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 (101.5), 56 μM (101.75), 100 μM (102), 177.8 μM (102.25) and 316 μM (102.5). 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
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
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
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.
RPE cells constitute a highly desirable source of cells for therapy in AMD (14, 15), but may also be used as an animal-free option for ocular drug discovery, toxicity screening and therapy (4, 35). hES-RPE and ARPE-19 are considered two suitable options for
The level of mRNA expression of RPE-markers was assessed by Real-time PCR and compared to primary human RPE cells from both fetal and adult tissues since it is known that RPE goes through significant maturation during life. Obtained results show that hES-RPE cells differentiated in our hands present an expression status highly similar to fetal RPE, but also intermediate compared to adult RPE, allowing us to classify such cells as “young RPE”. In an opposite direction, ARPE-19 mRNA profile was far different from both primary RPE cells, confirming that this cell line has lost important RPE markers, with the exception of ZO-1, under aforementioned conditions of cellular maintenance and expansion. As expected, hESC-RPE presented a lower expression of pluripotency genes, compared to undifferentiated counterparts.
Even though mRNA expression may be an important indication of cellular phenotype, protein and functional assays are important for phenotype validation. Therefore, the protein expression of MITF and BEST were assessed using immunofluorescence, and the expression of RPE-65 and CRLBP were assessed using western blotting. Both analysis confirmed the mRNA expression analysis, further supporting the notion that hESC-RPE were differentiated and presented an RPE phenotype.
The functionality of both hES-RPE and ARPE-19 was investigated according to the capacity of polarized VEGF synthesis and melanin content. Furthermore, cellular behavior of both cell samples was assessed when they were seeded on a surface that mimics the area where RPE cells are originally found in the ocular globe. Due to the fact that the changes in BM engendered by aging and AMD are complex and may not be fully reversible (38, 39), any source of RPE able to regenerate this area is desired. In this sense, not only BM adhesion may add a functional evidence to support their similarity compared to functional native RPE, but also reveals a possible suitability of those cells for AMD cellular therapy. SEM analysis showed that hES-RPE were able to resurface aged BM, in stark contrast to ARPE-19, which failed to attach, survive and proliferate on this surface.
Finally, the use of stem cell-derived RPE for drug screening constitutes a powerful approach for the development of new agents. Indeed, previous studies have been shown that the safety and efficacy can be enhanced by the use of relevant human cellular models, at the same time that the use of animals for such purpose can be reduced (27, 40) CsA, SRL, TAC, and LEF are drugs with known immunosuppressive properties, fully explored as corticosteroid-sparing agents for the treatment of many inflammation processes, including ocular diseases. However, these drugs had their use limited by the poor water solubility and severe side effects. Considering their intraocular administration, it becomes crucial to analyze their cytotoxicity, for instance, by evaluating each chemical in contact with the RPE cells. According to MTT assay results, ARPE-19 is more sensitive to the toxicity effects of tested drugs compared to hES-RPE. This behavior was observed in both MTT and CellTiter-BlueⓇ cell viability assays for the majority of the tested chemicals. Despite differences, both MTT and CellTiter Blue assays indicate a more “robust” phenotype of hES-RPE cells compared to ARPE-19.
In summary, we have highlighted how hES-RPE and ARPE-19 cells show different profiles
Supplementary data including one figure can be found with this article online at https://doi.org/10.15283/ijsc20094
ijsc-14-1-74-supple.pdfThe authors acknowledge the support from the Midwest Eyebank (includes eye banks in Illinois, Michigan, and New Jersey-USA) and for the financial support from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/Brazil), Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG/Brazil), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Pró-reitoria de Pesquisa da Universidade Federal de Minas Gerais.
The authors have no conflicting financial interest.
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