Animals

Non-pigmented and pigmented male rabbits were purchased from Jinan Xilingjiao Breeding Center (Shangdong, China) and housed and cared for in the animal facility of Shandong Eye Institute in accordance with the Principles of Laboratory Animal Care. Non-pigmented rabbits (3.5 kg, 6 months old) were used to establish the model of corneal endothelial dysfunction, while three-month-old non-pigmented and pigmented rabbits were used for primary rabbit RPE cells and CECs culture. All animal experiments followed the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, which is in accordance with the guidelines and regulations approved by the Ethics Committee of the Shandong Eye Institute (W202202220025).

Primary culture of rabbit RPE cells and CECs

Rabbits were euthanatized, and the eyeballs were stored at 4℃ for the subsequent culture of primary rabbit RPE cells and CECs. As previously described [37, 38], primary RPE cells were isolated and cultured for subsequent study. Briefly, the eyes were immersed in saline containing 400 U/mL gentamicin sulfate (CISEN Pharmaceutical Co., Shandong, China) for at least 30 min. Next, the intact corneas were separated and stored in Dulbecco’s modified Eagle’s medium (DMEM) (Corning, Manassas, VA, USA) for subsequent primary CEC culture. Excess tissues, including the lens, vitreous, and retina, were removed to obtain the optic cup, in which RPE cells were still attached on the inner side. Primary RPE cells were detached by 0.25% trypsin–EDTA (Sigma-Aldrich, Missouri, USA) for 1 h at 37 ℃, then gently isolated from Bruch’s membrane and triturated into a cell suspension. RPE cells were cultured in a medium consisting DMEM, 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), 1% penicillin/streptomycin (PS, Corning), 10 μM Y-27632 (Sigma-Aldrich), 10 μM nicotinamide (NAM, Sigma-Aldrich), and 1 μM SB431542 (Millipore, Boston, Massachusetts, USA) at 37 ℃ under 5% CO2. A total of about 1.6 × 104 RPE cells could be obtained from one single eye of rabbits, and 8 × 103 cells were seeded in one well of a 24-well plate. Cultured RPE cells were observed and checked microscopically every other day. Contaminant cells (such as fibroblasts) were removed mechanically. The cultures used for experimentation were confluent and exhibited a typical cobblestone-like morphology.

Primary rabbit CECs were cultured according to a previously reported method [39]. Briefly, Descemet’s membrane (DM) and corneal endothelium were separated under a microscope and cultured in DMEM supplemented with 10 μM Y-27632 overnight. Next, CECs were digested from DM with 0.6 U/mL of collagenase I (Sigma-Aldrich) for 1 h and cultured in a medium including DMEM, 10% FBS, 1% PS, 2 ng/mL human basic fibroblast growth factor (bFGF, R&D System, Minneapolis, USA), 1% insulin-transferrin-selenium (ITS, Gibco), 1 μM SB431542, and 10 μM Y-27632 at 37℃ under 5% CO2. Cells from two intact corneas were seeded in one well of a 12-well plate.

The morphologies of the primary RPE cells and CECs were observed using an inverted contrast phase microscope (Nikon TE 2000‐U, Nikon, Tokyo, Japan). Cells were subcultured when they reached confluence and cultivated primary cells at passage 2 were used for the following transplantation.

hESC culture and differentiation

The human embryonic stem cell line H1 was maintained in the serum-free mTeSR1 medium (StemCell Technologies, Vancouver, Canada) in plates coated with growth factor-reduced Matrigel (Corning) at 37 °C under 5% CO2. As previously described [40], RPE cells were derived from the hESCs. Briefly, hESCs (2 × 104 cells) were seeded on 1% Matrigel-coated dishes and cultured in mTeSR1 for 10 days. Then, cells were grown in a differentiated medium consisting of Dulbecco's modified Eagle medium/Nutrient Mixture F-12 (DMEM/F12, Invitrogen, Carlsbad, California, USA), 15% (vol/vol) KnockOut serum (Invitrogen), 2 mM glutamine (Invitrogen), 1 × nonessential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), and 1 × antibiotic–antimycotic (Invitrogen). Moreover, 10 mM NAM and 50 nM chetomin (CTM, Sigma-Aldrich) were added to the differentiated medium for the first 10–15 days of differentiation. After retention in DM only for more than three weeks, differentiated RPE cells were obtained. The morphologies of the hESCs and hESC-derived RPE cells were observed using a Nikon TE 2000‐U.

Intracameral cell transplantation and postoperative examinations

The rabbit corneal endothelial dysfunction model was performed as previously described [22, 41]. In short, rabbits were anesthetized with intramuscular ketamine hydrochloride (40 mg/kg, Gutian Pharmaceutical Co., Fujian, China) and intravenous pelltobarbitalum natricum (50 mg/kg, Sinopharm Chemical Reagent Co., Shanghai, China). The central corneal endothelium in the right eye of each rabbit was mechanically stripped from the DM using a 20-gauge soft silicone needle (Inami, Tokyo, Japan). Depending on the types of transplanted cells at the next stage, a curettage model of different sizes was adopted, a diameter of 7 mm was applied to primary RPE cells and CECs transplantation, and a diameter of 9 mm for hESC-derived RPE cells. Next, after irrigating the cell debris with saline, the surgeon injected heparin sodium (625 U/mL, Qianhong Bio-pharma Co., Changzhou, China) into the anterior chamber to reduce inflammation. Finally, the incision was sutured, and the model was established. The right eyes of corneal endothelial-damaged rabbits (n = 3) without cell injection were used as a negative control, and the right eyes of normal rabbits (n = 3) were used as a positive control.

Cells including primary rabbit RPE cells, CECs, and hESC-derived RPE cells were dissociated with Accutase (StemCell Technologies, Vancouver, Canada) for 15 min at 37 ℃ and then gently triturated into cell suspension. Next, 3 × 105 primary RPE cells (n = 3), 3 × 105 primary CECs (n = 3), and 8 × 105 hESC-derived RPE cells (n = 3) in 250 μL DMEM supplemented with 100 μM Y-27632 were injected into the anterior chamber immediately after closing the incision. The rabbits were still under anesthesia after surgery and were kept in the eye-down position on the operating table for 3 h to promote cell attachment. For postoperative care, all operative eyes were treated with topical medication for 14 days. Specifically, tobramycin and dexamethasone eye drops (Novartis, Basel, Switzerland) with 10 mM Y-27632 were administered four times a day. Meanwhile, local subconjunctival injections of a 1:1 mixture of 5 mg/mL dexamethasone sodium phosphate (CISEN Pharmaceutical Co.) and 0.5 mg/mL atropine sulfate (Kingyork Co., Tianjin, China) were administered once daily.

Corneal clarity, central corneal thickness, and intraocular pressure of operative eyes on days 1, 3, 7, 14, and 30 were monitored and measured using slit-lamp microscopy (SL-D7, Topcon, Tokyo, Japan), optical coherence tomography (OCT, Fremont, USA), and a tonometer (Tono-Pen AVIA, Reichert, NY, USA), respectively.

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)

The relative differential mRNA expressions of rabbit RPE cells and CECs were monitored by qRT-PCR analysis. Total RNA was extracted using the MiniBEST Universal RNA Extraction Kit (TakaRa, Tokyo, Japan). Next, complementary DNA (cDNA) was synthesized by HiScript III RT SuperMix for qPCR (Vazyme, Nanjing, China), and qRT-PCR was performed using SYBR Green qPCR Master Mix (Vazyme) according to the manufacturer’s protocol. The quantified data were analyzed and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers used in the qRT-PCR are listed in Table 1.

Table 1 Primer sequences for quantitative reverse transcription-polymerase chain reactionImmunofluorescent staining

Cultured primary rabbit RPE cells, CECs, and hESC-derived RPE cells were fixed with 4% paraformaldehyde (PFA) (Biosharp, Anhui, China) for 10 min. After incubating in 0.3% Triton X-100 (Beyotime Biotechnology, Shanghai, China) for 5–15 min, all species of cells were incubated with 5% bovine serum albumin (BSA) (Boster Biological Technology, Wuhan, China) for 1 h to block nonspecific binding sites at room temperature then treated with primary antibodies (Table 2) at 4 ℃ overnight. After incubating with Alexa Fluor 488- or 594-conjugated secondary antibodies (Invitrogen, Carlsbad, California, USA) (Table 2) for 1 h at room temperature, nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (Beyotime Biotechnology), and fluorescence images were then captured using the Echo Revolve-100-G (ECHO, San Diego, California, USA).

Table 2 Antibodies for immunofluorescence staining

Normal rabbit eyeballs were obtained post-euthanasia for in situ immunofluorescent staining of the RPE and corneal endothelium. Briefly, the cornea was removed from the eyeball and fixed with 4% PFA for 12 min then stored at 4 ℃ until further use. The eye cup was obtained after removing the segments of the eye, including the iris, lens, and vitreous body. After incubating with 4% PFA for 30 min, the neurosensory retina and sclera were removed while the RPE-Buch’s membrane-choriocapillaris complex (RBCC) was acquired and stored at 4 ℃ as previously described [42]. Subsequently, in situ rabbit cornea and RBCC were permeabilized with 0.5% Triton X-100 for 5–15 min. After washing rabbit tissues with PBS three times, they were blocked in 2.5% BSA for 1 h at room temperature and incubated with primary antibodies (Table 2) overnight at 4 ℃. Both the rabbit cornea and RBCC were washed to remove excess primary antibodies the next day and then incubated with secondary antibodies (Table 2) for 1 h at room temperature. The nuclei were stained with DAPI, and the fluorescence images were observed using a laser scanning confocal microscopy (LSM 800, Zeiss, Jena, Germany).

For the immunofluorescent staining of transplanted hESC-derived RPE cells, primary rabbit RPE cells, and CECs, the corneas with the transplanted cells mentioned above were obtained after euthanasia and fixed with 4% PFA for 12 min. The following staining procedures were consistent with those of rabbit corneal endothelium in situ.

Na+/K+-ATPase activity

Primary RPE cells and CECs were cultured and collected at passage 2 to determine their Na+/K+-ATPase activities. Their Na+/K+-ATPase activities were tested using the indicated Na+/K+-ATPase activity kits (Solarbio, Beijing, China) according to the manufacturer’s protocol.

Cell permeability

Primary rabbit RPE cells and CECs at passage 1 were implanted in the apical side of the filter (Transwell, Corning) and their cell permeabilities were monitored using the horseradish peroxidase (HRP) tracing technique when the cells reached confluence for 5 days. HRP (50 μg/mL) in 200 μL culture medium was added to the apical side of the filter, while basal compartment medium was replaced with 500 μL culture medium without tracer. Cells were incubated at 37 ℃, and then basal compartment medium was collected at 5 min, 10 min, 15 min, and 30 min. Next, 195 μL o-phenylenediamine (Sigma-Aldrich) was reacted with the collected culture medium with tracer for 1 min, and 25 μL H2SO4 were immediately added to terminate the reaction. The color in each well changed from blue to yellow. Tracers were quantified by OD at 492 nm using a microtiter plate reader, and permeability was calculated as Flux = (X)B/(Y)i/A where (X)B represented counts in the basal chamber (μg), (Y)i was the initial concentration in the apical chamber (μg/mL), and A was the area of the filter (cm2).

Electron microscopy

The RBCC and rabbit corneal endothelium were examined by scanning electron microscopy and transmission electron microscopy, respectively. The RBCC and rabbit corneal endothelium were immersed and preserved in fixative (Servicebio, Wuhan, China) at 4 ℃ and postfixed with 1% OsO4 in 0.1 M PB (pH 7.4) for 2 h at room temperature, avoiding light. After rinsing in 0.1 M PB (pH 7.4) three times, the tissues were dehydrated in a graded ethanol (Sinopharm Group Chemical Reagent Co. Ltd, China) series. For scanning electron microscopy preparation, the tissues were dried with a critical point dryer (Quorum, UK). Specimens were attached to metallic stubs using carbon stickers and sputter-coated with gold for 30 s before examination with scanning electron microscopy (Hitachi, Japan). For transmission electron microscopy preparation, the tissues were embedded in resin (EMBed 812). Ultrathin sections (60–80 nm thin) were fished out onto cuprum grids with formvar film and were stained with 2% uranium acetate saturated alcohol solution for 8 min as well as 2.6% lead citrate for 8 min. After drying overnight at room temperature, the cuprum grids were observed under transmission electron microscopy (Hitachi), and images were taken.

Enzyme linked immunosorbent assay (ELISA)

The anterior chamber humors of the hESC-RPE cell transplanted rabbits and non-injected corneal endothelial-damaged rabbits were collected on day 30 after surgery, and the concentrations of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF, Kete, Jiangsu, China) were quantified using the indicated ELISA kits according to the manufacturer’s instructions.

Statistical analysis

All data obtained from at least three independent experiments were expressed as the mean ± standard error of the mean for the values. A student’s t-test or one-way ANOVA was used to compare the mean values between the groups using SPSS (v.19.0, Chicago, IL, USA), while P < 0.05 and P < 0.01 were considered as the significance thresholds.