Int J Stem Cells.  2021 Feb;14(1):74-84. 10.15283/ijsc20094.

Human Stem Cell-Derived Retinal Pigment Epithelial Cells as a Model for Drug Screening and Pre-Clinical Assays Compared to ARPE-19 Cell Line

• Oliveira CR
• Paiva MRBd
• Ribeiro MCS
• Andrade GF
• Carvalho JL
• Gomes DA
• Nehemy M
• Fialho SL
• Silva-Cunha A
• Góes AMd

• KMID: 2513084
• DOI: http://doi.org/10.15283/ijsc20094

Abstract

Background and Objectives
Eye diseases have a high socioeconomic impact on society and may be one of the fields in which most stem cell-related scientific accomplishments have been achieved recently. In this context, human Pluripotent Stem Cell (hPSC) technology arises as an important tool to produce and study human Embryonic Stem cell derived-Retinal Pigmented Epithelial Cells (hES-RPE) for several applications, such as cell therapy, disease modeling, and drug screening. The use of this technology in pre-clinical phases attends to the overall population desire for animal-free product development. Here, we aimed to compare hES-RPE cells with ARPE-19, one of the most commonly used retinal pigmented epithelial immortalized cell lines.
Methods and Results
Functional, cellular and molecular data obtained suggest that hES-RPE cells more closely resembles native RPEs compared to ARPE-19. Furthermore, hES-RPE revealed an interesting robustness when cultured on human Bruch’s membrane explants and after exposure to Cyclosporine (CSA), Sirolimus (SRL), Tacrolimus (TAC), Leflunomide (LEF) and Teriflunomide (TER). On these conditions, hES-RPE cells were able to survive at higher drug concentrations, while ARPE-19 cell line was more susceptible to cell death.
Conclusions
Therefore, hES-RPEs seem to have the ability to incur a broader range of RPE functions than ARPE-19 and should be more thoroughly explored for drug screening.

Keyword

ARPE-19; Human embryonic stem cell derived-retinal pigmented epithelial cells; In vitro toxicology; In vitro model

Figure

• 

  Fig. 1 hES-RPE differentiation and experimental design. (A) hES-RPE were differentiated by proliferation and multilayer formation of hES cells and removal of bFGF of the medium. (B) hES-RPE and ARPE-19 were first characterized according to mRNA expression of RPE markers, then according to Bruch’s membrane resurface ability. Finally, hES-RPE and ARPE-19 were used for cytotoxicity assays.

• 

  Fig. 2 mRNA and protein expression of RPE markers. (A) The mRNA expression of RPE-65, BEST, CRLBP, MITF, PEDF, and ZO-1 was evaluated by quantitative real-time polymerase chain reaction. mRNAs were quantified from hES-RPE, ARPE-19, fetal RPE, and adult RPE normalized to the geometric mean of a housekeeping gene (18s rRNA). (B) The mRNA expression of pluripotency (OCT4 and NANOG), neuroectoderm (PAX6) and RPE markers (MITF, CRLBP, BEST, RPE-65, PEDF and ZO-1) was also compared between undifferentiated hESCs and hESC-RPE. As expected, compared to undifferentiated H1, differentiated cells at passage 2 presented lower expression of pluripotency genes, and higher expression of neuroectoderm and RPE differentiation markers. (C) Protein expression of BEST and MITF was evaluated in p2 hESC-RPE cells by immunofluorescence. As expected, BEST presented membrane localization, and MITF, nuclear localization. (D) Western Blotting analysis of RPE-65 and CRLBP expression by p1, p2 and p3 hESC-RPE cells. Similar to fetal RPE, hESC-RPE cells from all the analysed passages presented detectable expression of both RPE markers. Bars represent standard error of the mean. Abbreviations: hES-RPE, RPE derived from human embryonic stem cells; fRPE, fetal RPE.

• 

  Fig. 3 Functional analysis of hESC-RPE cells. (A) Melanin content of passage 2 hESC-RPE cells in different times of culture. (B) Polarized VEGF synthesis of hESC-RPE cells. As depicted, hESC-RPE cells preferentially secrete VEGF in the basal side. Bars represent standard error of the mean. Abbreviations: hES-RPE, RPE derived from human embryonic stem cells; BM, human Bruch’s membrane.

• 

  Fig. 4 Analysis of RPE cells’ ability to resurface aged human Bruch’s membrane (BM) explants. (A, B) SEM micrography of the superficial structure of the BM’s illustrating exposition of the basement membrane layer. (C, D) ARPE-19 cells seeded and cultured on BM’s were not able to proliferate and the explant was only partially resurfaced; (E, F) hES- RPE cells seeded and cultured on BM’s were able to survive, proliferate and virtually resurface the whole explant, with small flaws (narrow arrows). hES-RPE formed a cell layer with a morphology that resembles the hexagonal character of native fetal RPE (wide arrows). *Represents an artifact occurred during manipulation of the sample.

• 

  Fig. 5 Drug cytotoxicity analysis of sirolimus, leflunomide, and teriflunomide evaluated by MTT Tetrazolium Assay (I) and Resazurin Reduction Assay (CellTiter-BlueⓇ Cell Viability Assay) (II). Cyclosporin was not cytotoxic for both cell types in the analyzed doses, however, showed a reduction of cell viability greater than 50% for hES-RPE at the dose of 100 μM. Sirolimus (IB and IIB) showed higher cytotoxicity than leflunomide and its active metabolite, teriflunomide. TAC treatment (IC and IIC) showed cytotoxic for ARPE-19 at dose evaluated 100 μM (102) while it was cytotoxic for hES-ARPE at dose evaluated 176 μM (102.25). Leflunomide treatment (ID and IID) was cytotoxic for ARPE-19 at the highest dose evaluated (178 μM, 102.25) while it was not cytotoxic for hES-ARPE cells at this concentration. Teriflunomide treatment (IE and IIE), however, showed cytotoxicity for both cell types with the dose of 178 μM (102.25), and at 100 μM (102) ARPE-19 cells were also susceptible, but not hES-RPE cells. *significant difference comparing hES-RPE control and hES-RPE treatments; #significant difference comparing ARPE-19 control and ARPE-19 treatments; ＋significant difference comparing hES-RPE and ARPE-19 at same concentration.

Reference

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