Animals

Pigmented rabbits that weighed between 1.5 kg and 2.0 kg were used in this study (Department of Physiology, Isfahan University, Iran). All proceedings concerning animals used were performed in accordance with the Ethical Committee at Royan Institute. Pigmented rabbits were sacrificed by an overdose of ketamine and xylazine. After enucleating eyes from anesthetized rabbits, extra ocular tissues were cleaned. Intact globes were washed in Ca₂+ and Mg₂+– free phosphate buffered saline supplemented with penicillin/streptomycin.

Then, globes were incubated in 2% dispase (Gibco, 17105-041) for 20 min. Cornea-iris complex cut-off just 3 mm posterior to the limbus. Vitreous and anterior segment were removed. The posterior eye cup was dissected by four incisions. After incubation of posterior segment in Dubecco's Modified Eagle's Medium (DMEM)/F12 supplemented with 10% fetal bovine serum for 2 h, the RPE layer was peeled off in sheet and used for co-culture.

Culture of hESCs

The Royan H5,[16] hESC line was obtained from Royan Institute. The cells were cultured on martrigel under feeder-free culture condition in the presence of media previously described.[17] The media were changed every other day, for 7 days.

Tissue culture

After 7 days, the differentiated cells in the center of colony mechanically discarded and the undifferentiated cells of hESCs, which usually located in the peripheral part was induced to neural ectoderm in the presence of media containing noggin (1 ng/mL; R and D, 1976-NG), human recombinant Dkk-1 (1 ng/mL; R and D, 5439-DK/CF), and human recombinant insulin-like growth factor-1 ([IGF]-1, 5 ng/mL; R and D, 291-GI) in DMEM-F12 medium supplemented with 10% knockout serum replacement, 0.1 mM non-essential amino acids, 2 mM L-glutamine, and 1% B27 (Gibco, 17504-044), for 2 days. On the 3^rd day, the cells were cultured in the presence of retinal determination (RD) medium that consisted of DMEM: F12 supplemented with 1% B27, 2% N2 (Gibco, 17502-048), 10 ng/mL noggin, 10 ng/mL Dkk-1, 10 ng/mL IGF-1, and 5 ng/mL bFGF as previously described.[18] The medium was renewed every other day up to 2 weeks to form the neural tube (NT)-like structures. On day 16, NTs were manually dissociated and replanted on 1 mg/mL laminin and 15 mg/mL poly-L-ornithine (both from Sigma-Aldrich)-coated 6-well tissue culture plates (TPP, 92406) in the same medium (20-30 NTs per well).

In vitro explant co-culture experiments

To evaluate the differentiation of RPCs, co-cultures of RPE sheet and these cells were obtained by following three procedures. One day after replanting of the dissociated NTs on day 17, by two methods (Indirect), the isolated RPE sheet was cultured on filter insert 0.4-mm (Millicell-CMPICMO3050) and transferred into six well plates that included the dissociated NTs for either 1 week or 2 weeks in the presence of RD medium. In the third method, RPE sheet was seeded over the RPCs (direct) for 2 weeks in RD medium. The cells from all groups were cultured for an additional period of 10 days in neurobasal medium (Invitrogen, 21-103) supplemented with 2% N2. Half of medium was renewed every other day.

Immunocytochemistry staining

Cell cultures were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), followed by permeation with Triton X-100 (0.1%) for 15 min. To block non-specific sites, we used 10 mg/ml bovine serum albumin. Retinal cells were identified by the antibodies listed in Table 1. After exposing the cells overnight to primary antibodies, the cells were washed 3-5 times with PBS for 5 min and exposed 1 h with either fluorescein isothiocyanate-or TRITC-conjugated secondary antibodies, and nuclei were stained with DAPI (4’,6-diamidino-2-phenylindole) (Sigma, D8417).

Table 1. Antibodies used in this study

Reverse transcription-polymerase chain reaction for gene expression analysis

The total RNA was extracted from the differentiated cells and digested with deoxyribonuclease I (Fermentas, EN0521). Standard RT was performed using random hexamer primers, and RevertAid™ First strand complementary deoxyribonucleic acid (Fermentas, K1622). PCR reaction was followed using SmarTaq polymerase and specific primers shown in supplementary Table 2. Amplification conditions were as follows: initial denaturation at 94°C for 5 min was followed by 35 cycles of denaturation at 94°C for 30 s, annealing for 45 s and extension for 45 s at 72°C and a final polymerization at 72°C for 10 min. PCRs were performed in triplicate. PCR products were analyzed by gel electrophoresis on 1.7 or 2% agarose and stained with ethidium bromide (10 μg/ml). They were visualized and photographed on an ultraviolet transilluminator.

Table 2. Primer sequences and conditions of RT-PCR

Characterization of RPE cells

Examination of RPE culture sheets by phase-contrast microscopy showed a monolayer of uniform hexagonal morphology of RPE cells with melanine [Figure 1]. Staining with anti-ZO-l, tight junction protein and cytokeratin 18, an intermediate protein, demonstrated the expression of these proteins in the RPE sheets [Figure 2]. Reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed the expression of natriuretic peptide receptor-A, a rabbit RPE marker, in these cells, even after 14 days [Figure 3]. In addition, one-way ANOVA analysis of MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) results revealed no significant different between the viability of RPE cells in first and 14th days (mean OD: 1.435 ± 0.32 and 1.542 ± 0.247) respectively.

[Figure ID: F1] Figure 1. Phase contrast photomicrographs of retinal pigment epithelium isolated cells that showed morphology of these cells. Scale bar: 50 μm

[Figure ID: F2] Figure 2. Isolated retinal pigment epithelium (RPE) in vitro and experimental design. (a) isolated RPE expressed intermediate cytokeratin-18 protein and (b) tight junction protein ZO-1. Scale bar: 100 μm

[Figure ID: F3] Figure 3. Reverse transcription chain reaction analysis showing the expression of rabbit retinal pigment epithelium

Differentiation of hESCs into RPCs

To generate RPCs, the undifferentiated feeder-free hESCs were treated with noggin, Dkk-1 and IGF-1 for 2 days and then switched to RD medium for 2 weeks without embryoid body formation in adherent conditions. Subsequently, the neuro epithelium and NTs emerged from hESCs. These data demonstrate that a large fraction of the hESCs in these cultures are characteristic of retinal progenitors (RPs) [Figure 4]. Quantification of immunostaining showed that 79% ± 1.6% of differentiated cells were positive for Rx. In addition, 78.3 ± 1.8% of the cells expressed Pax6 and 86.2 ± 1.6% of the differentiated cells were positive for Otx2.

[Figure ID: F4] Figure 4. Quantitative immunofluorescence staining showed high expression of RP markers (a) Pax6 (b) Otx2 (c) RX. Scale bar: 200 μm

Co-culture of RPCs with retinal pigment epithelium

To assess whether the close contact of RPE cells could be effective for the generation of neural photoreceptors, we co-cultured the RPE with hES cell-derived progenitors on 0.4 mm filters for one and 2 weeks or without filters in the presence of RD medium. Before the co-culture, we evaluated the viability and identity of RPE cells after 14 days. The observation of microscopic images suggests that photoreceptor markers are better expressed in use with filter for 2 weeks and direct co-culture as compared to control group [Figures 5 and 6]. In the indirect co-culture, after 1 week, it did not improve the photoreceptor differentiation (data not shown). Direct co-culture expressed some mature photoreceptor markers such as arrestin in compared to the indirect co-culture for 2 weeks and control group. There were no differences between the expressions of other photoreceptor markers [Figure 7].

[Figure ID: F5] Figure 5. Immunofluorescence staining showed expression of S-opsin (a) and rhodopsin (b) in retinal progenitors a-derived retinal cells in direct co-culture. Scale bar: 200 μm

[Figure ID: F6] Figure 6. Expression of S-opsin (a) and rhodopsin (b) in retinal progenitors-derived retinal cells in indirect co-culture after 2 weeks. Scale bar: Respectivly 200 and 100 μm

[Figure ID: F7] Figure 7. Reverse transcription chain reaction analysis demonstrated expression of early photoreceptor markers in direct and indirect co-culture and late photoreceptor marker like arrest in in direct co-culture