Culture of hiPSCs
The hiPSCs were a gift from the Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences. These hiPSCs were reprogrammed from human umbilical cord mesenchymal cells using methods described in a previous report . hiPSCs were cultured in 1% Matrigel (BD Biosciences, USA) coated dishes at 37 °C, 5% CO2 and refreshed daily with mTeSR1 medium (StemCell Technologies Inc., Canada). They were passaged once every 6 days with 0.25% EDTA (Sigma, USA) and were then seeded into 6-well dishes at a ratio of 1:6. They were supplemented with 10 µM Y-27632 (Sigma) on the first day of passaging. For generating dissociated hiPSCs, the harvested clonal hiPSCs were pipetted approximately 30–50 times and then were filtered through a 40 µm cell strainer (BD). The dissociated hiPSCs (1 × 106 cells/well) were plated into 6-well dishes. Half-exchange mTeSR1 medium (HM) was used for further culturing of the dissociated hiPSCs based on the non-colony type monolayer (NCM) .
Isolation and culture of adult RPE cells
Human eyes were obtained from six male donors after informed consent at the mean age of 45 ± 5 years old. The study was approved by the Human Research and Ethical Committee of Jinan University and the procurement and use of human tissues were in compliance with the Declaration of Helsinki. Human adult RPE cells were isolated and cultured as described in a previous report . Briefly, RPE cells were isolated from the posterior section of the eyeballs using 0.25% EDTA-trypsinase (Gibco, USA) and harvested by centrifugation at 400 × g for 5 min. hRPE cells were cultured in RPE medium that consisted of high-glucose Dulbecco’s modified Eagle’s medium (HG-DMEM, Gibco), 10% FBS (Gibco), 100 U/mL penicillin and 100 mg/mL streptomycin (P/S, Gibco) at 37 °C and 5% CO2. After reaching 100% confluence, the RPE cells were passaged and seeded in 6-well plates with a 1% Matrigel coating (Sigma). The medium containing 100 ng/ml Activin A (R&D Systems, USA) was changed every 3 days.
Differentiation of NCM dissociated hiPSC into RPE cells
The point at which dissociated hiPSCs expanded to 100% confluence in HM was defined as day − 2, and from day − 2 to 0, hiPSCs were cultured in E7 medium without FGF2 but with 10 µM Y-27632. Prior to the beginning of differentiation, dissociated hiPSCs were cultured in proneural medium containing DMEM/F12 (Gibco), 1% nonessential amino acids (NEAA, Invitrogen, USA), and 1% N2 (Invitrogen). From 0–2 days, 10 ng/mL IGF-1 (R&D Systems), 50 ng/mL Noggin (R&D Systems), 10 ng/mL Dkk-1 (R&D Systems), and 10 mM nicotinamide (NIC, Sigma) were added into the proneural medium. From 2–4 days, 10 ng/mL IGF-1, 10 ng/mL Noggin, 10 ng/mL Dkk-1, 10 mM NIC, and 5 ng/mL bFGF were added to the proneural medium. From 4–6 days, 10 ng/mL IGF-1, 10 ng/mL Dkk-1, and 100 ng/mL Activin A were added to the proneural medium. From 6–14 days, 100 ng/mL Activin A and 10 µM SU5402 (EMD Millipore, Germany) were added to the proneural medium. Then, the differentiated cells were mechanically enriched by scraping away cells with non RPE-like (spindle) morphology, and the remaining RPE-like (epithelioid-like) cells were passaged using 0.25% EDTA and seeded into 1% Matrigel coating dishes. From 14–20 days, the enriched cells were cultured in enrichment medium containing HG-DMEM (Gibco), 1% FBS (Gibco), 100 ng/mL Activin A, 1 × sodium pyruvate, and 1 × GlutaMAX (Invitrogen). From 20–30 days, these enriched cells were passaged again as passage 1 (P1) and cultured in RPE medium.
Gene expression analysis
Total RNA from the cells was isolated using a Tissue RNA Miniprep Kit (Biomega, China). The cDNAs were synthesized and used for reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR products were examined after electrophoresis on 2% agarose gels. Gels were scanned for further analysis. For quantification of gene transcripts, cDNAs were first denatured at 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s and 58 °C for 30 s. The relative expression of the genes was normalized against GAPDH, and quantification was performed using the comparative Ct (2− ΔΔCt) method. The primer sequences are shown in Table 1.
Samples were fixed using 4% paraformaldehyde and permeabilized by 0.1% Triton X-100 (Sigma). Then, they were incubated with isotype control or primary antibodies as shown in Table 2 at 4 °C for 30 min. Primary and isotype control antibodies were labeled with fluorophore-conjugated secondary antibodies at 4 °C for 30 min. The labeled samples were detected by a flow cytometry analyzer (BD, USA).
An immunofluorescence assay was used to identify the hiPSC-RPE cells as described in a previous report . Briefly, paraformaldehyde fixed cells were permeabilized with 0.1% Triton X-100 and incubated with 3% (w/v) BSA for blocking. Cells were then incubated with primary antibodies as shown in Table 2 overnight at 4 °C. On the second day, the cells were washed twice with PBS and then incubated with FITC-conjugated anti-mouse, FITC-conjugated anti-rabbit, Cy3-conjugated anti-mouse, or Cy3-conjugated anti-rabbit IgG secondary antibodies (1:1000, Bioword, USA) at room temperature for 60 min. Cells were rinsed 3 times with PBS and stained with DAPI (Sigma) before examination by a fluorescence microscope (OLYMPUS, Japan).
Fluorescein leakage test
A fluorescein leakage test (FLT) was used to assess the barrier function of the epithelial cells as described in a previous report . First, 200 µL of cells (1 × 105 cells/mL) were seeded in 6-transwell inserts (Millipore, USA) and incubated at 37 °C and 5% CO2. After reaching 100% confluence, the cells were fixed with 4% paraformaldehyde and 500 µL of Na-fluorescein solution (10 µg/mL, Sigma) was added into each insert before incubation for the designated time (0, 4, 12, 24, and 36 h) to allow solution leakage into the bottom wells. The values of fluorescein leakage into the wells were measured using a microplate spectrophotometer (VSERSA Max, USA) at 485 nm excitation and 530 nm emission wavelengths. The fluorescein leakage through the inserts without cells was set as 100% (maximum leakage). The fluorescein leakage test (FLT) values were calculated as the amount of fluorescein leakage from hRPE and hiPSC-RPE cells compared to the corresponding maximum leakage.
Transepithelial electrical resistance (TEER) assay
A TEER assay was used to assess the dynamic barrier function of the epithelioid cells . Cells were seeded into 24-transwell inserts at 1 × 104 cells/insert. After reaching 100% confluence on day 7, the dynamic barrier of the cells was determined through measuring TEER across the cell monolayer using Millicell-ERS-2 (Millipore, Temecula, USA). The value of TEER was calculated per the following equation:
TEER (Ω cm2) = (Rtotal-Rinsert w/o Matrigel) × A
Rtotal is the resistance measured (Ω), Rinsert w/o Matrigel (Ω) is the resistance of the insert with or without 1% Matrigel coating, and A is the membrane area (cm2) of the insert.
Atomic force microscopy observation
Atomic force microscopy (AFM) was used to observe the ultrastructure of cells as described in a previous report . Cells were fixed with 4% paraformaldehyde for 10 min and dried at room temperature before imaging. The curvature radius of the AFM tips was 10 nm. The spring constant was 20–50 N/m with a resonance frequency of 278–317 kHZ. The scanning speed was kept at 0.5 Hz. The ultrastructure of the cells was measured in contact mode. The data analysis was performed using Nanoscope Analysis Software (Thermo Microscopes Proscan Image Processing Software Version 2.1, USA).
POS phagocytosis assay
The photoreceptor outer segment (POS) was isolated as described in a previous report . Briefly, the retinas of porcine eyeballs were collected and agitated in KCl buffer (0.5 mM CaCl2, 1 mM MgCl2, 0.3 M KCl, and 10 mM HEPES) with 48% w/v sucrose at pH 7.0, and then were centrifuged at 5000 × g for 5 min. The supernatant containing the POS was filtered using sterile gauze, diluted 1:1 with KCl buffer without sucrose, and centrifuged at 4000 × g for 10 min. The isolated POS were then resuspended in 1 mL of PBS and were labeled with FITC (Sigma-Aldrich) at room temperature for 1 h. The labeled FITC-POS were then rinsed and resuspended using HG-DMEM medium with 5% sucrose. Cells were incubated with FITC-POS at 37 °C and 5% CO2 for 2 h. Last, immunofluorescence was performed using mouse monoclonal antibody ZO-1 and DAPI, and then they were examined under an inverted fluorescence microscope.
Z-stack confocal microscopy polarized membrane observation
Z-stack confocal microscopy was used to observe the polarized membrane of the cells. First, 200 µL of cells (1 × 106 cells/mL) were seeded into 6-well dishes and incubated at 37 °C and 5% CO2 for 5 days. Immunofluorescence was conducted with the mouse monoclonal antibody ZO-1 and DAPI and the stained cells were examined under a confocal microscope (LSM 510 META; Zeiss, Thornwood, USA).
Cells were washed using cold PBS and lysed using RAPI (Beyotime Biotechnology, China). A total of 50 µg of protein was electrophoresed on 10% SDS-PAGE gels and then transferred to polyvinylidene fluoride membranes (PVDF, Sigma) and blocked using 5% fat-free milk. Then, the membranes were incubated with primary antibodies as shown in Table 2 at 4 °C overnight. The membranes were washed 5 times with TBST and incubated with HRP-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (1:3000, Bioword) at room temperature for 2 h. Bands were visualized with enhanced chemiluminescence (ECL, Pierce, USA).
Production of agarose micro multiwell dishes and hiPSC-RPE cell spheroids
A volume of 500 µL liquid solution of 2% (g/mL) agarose was pipetted into an eighty-one well silicone micro-mold (Micro Tissues Inc., CA, USA). After solidification, the microwell agarose mold was removed using sterilized forceps. The agarose mold was placed in 6-well dishes. Then, 200 µL of cell suspension containing approximately 2 × 105 cells was carefully pipetted into the microwell plate and incubated at 37 °C and 5% CO2. The medium was changed every 2 days. Cell spheroids (approximately 80 µm diameter, 5 × 103 cells/spheroid) were formed after 3 days.
Preparation of decellularized corneal matrix (DCM)
The lamellar corneal matrix (100 µm thickness) was excised from porcine eyeballs using a microkeratome (Kangming, China) and was rinsed 3 times with PBS. The excised lamellar corneal matrix was treated with 0.25% EDTA-trypsinase (Invitrogen) at 37 °C for 30 min, and then was fixed with 4% paraformaldehyde for 1 day at 4 °C. It was then treated with 0.8% SDS (Sigma) solution at − 80 °C for 30 min and then was transferred to a 37 °C shaking table (350 rpm) for 1 h. It was then rinsed 3 times with PBS and preserved in 100% glycerol at 4 °C as decellularized corneal matrix (DCM). Before the seeding of cells, the DCM was washed 3 times with PBS containing P/S solution and sterilized under ultraviolet light for 2 h.
Seeding the cell spheroids on the DCM
To test whether cell spheroids in the biomimetic microenvironment could grow well, an in vitro simulation experiment of seeding cell spheroids onto DCM was conducted as described previously . Briefly, cell spheroids were seeded on the DCM. The medium containing 10 µM Y-27632 was changed every 3 days. Viable cell staining with Calcein AM was used for better observation of the cell spheroids on the DCM under a fluorescent microscope. The adherent growing area of the cell spheroid periphery stained by a Live-Dead Cell Staining Kit (Biotium, USA) was measured using ImageJ on days 7 and 14, respectively.
Frozen tissue sections
Tissue samples were mounted using tissue freezing medium (SAKURA Tissue-Tek, USA) and placed at − 80 °C until frozen. The frozen tissues were sectioned at a thickness of 15 µm using a cryo-microtome (Thermo Fisher, USA) as described in a previous report . Sections were placed on one side of microscope slides (SAKURA Tissue-Tek, USA). All of the sections were fixed with 4% paraformaldehyde for 15 min. Some sections were incubated for 15 min with DAPI for nuclear staining and examined with an inverted fluorescence microscope. Other sections were used for hematoxylin-eosin (H&E) staining and were imaged using an inverted microscope.
Live/dead assay of Calcein AM and EthD-III double staining
Calcein AM and EthD-III double staining (Molecular Probes, USA) was performed as described in a previous report . Briefly, a standard working solution containing 2 µM Calcein-AM and 4 µM EthD-III was prepared. Cells were incubated with the standard working solution at room temperature for 40 min and were then imaged under an inverted fluorescence microscope.
SA-β-Gal activity assay
SA-β-Gal activity was detected using a Cellular Senescence Assay Kit (Beyotime Biotechnology, China) follow the manufacturer’s instructions. We used spheroid or monolayer adherent cells on day 7 and 14 to measure SA-β-Gal activity. Briefly, after reaching 100% confluence, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature and were then incubated in a staining solution overnight at 37 °C. On the next day, the stained cells were washed with PBS and observed under an inverted microscope. A blue color indicated the presence of SA-β-Gal activity. The intensity of the SA-β-Gal activity was calculated using ImageJ software.
Tagging hiPSC-RPE cells with PKH26
The standard protocol was performed as described on the PKH26 Product Information Sheet (MINI2, Sigma). Briefly, a suspension containing 2 × 107 cells was centrifuged (400 × g, 5 min) and washed once using fresh medium without serum. After centrifuging, the cells were resuspended in 1 mL of Diluent C. Dye Solution (4 × 10− 6 M) was prepared by adding 4 µL of PKH26 ethanolic dye solution into 1 mL of Diluent C. Then, 1 mL of Dye Solution was rapidly added to the cell suspension. The final concentration after mixing was 2 × 10− 6 M PKH26 with 1 × 107 cells/well. The mixing suspension was incubated with periodic mixing at room temperature for 5 min. The staining was stopped by adding an equal volume (2 mL) of serum. Then, the suspension was centrifuged at 400 × g for 10 min and washed 3 times. Finally, cells tagged with PKH26 were used for injection.
RPE degeneration chinchilla rabbit model
Rabbits were weighed on an electronic scale and then injected with 1% NaIO3 (40 mg/kg, Sigma) via the ear marginal vein. After one week, the rabbits were injected with 1% NaIO3 (40 mg/kg) again. The next week, they could be used as an RPE degeneration model for cell transplantation.
Preliminary test of hiPSC-RPE cell spheroids in vivo
Animal experiments were approved by the Institutional Animal Care and Use Committee of Jinan University, and animal procedures were conducted following the guidelines of the US National Institutes of Health. Twelve six-month-old chinchilla rabbits with a weight of 1–2 kg were raised in a 12 h dark/light cycle, temperature at 23 ± 2 °C, and relative humidity of 45–55%. Water and food were changed every day. These chinchilla rabbits were randomly separated into four groups: control group (n = 3, 6 eyes), which received no treatment; Na2IO3 group (n = 3, 6 eyes), which received Na2IO3 treatment; Na2IO3 + PBS group (n = 6, 6 right eyes), which received Na2IO3 treatment and PBS injection; and the Na2IO3 + hiPSC-RPE group (n = 6, 6 left eyes), which received Na2IO3 treatment and hiPSC-RPE cell injection. Water containing 210 mg/L cyclosporin A (Sigma) and prednisone were given to these rabbits throughout the experiment to prevent allograft rejection. For PBS or hiPSC-PRE cell injection, the model rabbits were anesthetized with pentobarbital sodium (25 mg/kg, Sigma) and chlorpromazine (5 mg/kg, Sigma). The pupil was dilated using tropicamide (Alcon, Canada) and the eye lid was kept open using a lid speculum. Cell transplantation was performed under a surgical microscope (Ocular Instruments, China) . For subretinal injection, the peritomy was made 2.0 mm posterior to the limbus in the superotemporal quadrant of each eyeball. A sideport knife Beaver blade (BD) was used to make a longitudinal triangular scleral incision starting 2 mm away from the limbus at about the 5° axis toward the choroid until minimal blood reflux appeared. At this point an additional tract through the choroid toward the RPE layer was created using a 30-gauge needle. The hiPSC-RPE cell spheroids labeled with PKH26 were suspended in PBS containing 10 µM Y-27632, and then 10 µL of cell spheroids (approximately 1 × 105 cells) at a density of 20 cell spheroids/µL were slowly injected through the scleral tunnel using a 50 µL Hamilton blunt syringe with a 30-gauge needle (BD), then the syringe was immediately pulled back.
Rabbits were sacrificed with an overdose of sodium pentobarbital. Their eyeballs were removed and fixed in 4% paraformaldehyde overnight at 4 °C. Samples were dehydrated with a graded series of ethanol and xylene and subsequently were embedded using paraffin wax . The paraffin sections (5 mm) were cut and dewaxed in water. After antigen repair, these sections were fixed with 4% paraformaldehyde for 15 min and washed 3 times with PBS. They were permeabilized with 0.1% Triton X-100 for 15 min at room temperature and were blocked with 3% (w/v) BSA (Sigma) at room temperature for 1 h. Then, they were incubated with primary antibodies as shown in Table 2 for 2 h at room temperature followed by FITC-conjugated secondary antibody (1:1000, Bioword) for 1 h at room temperature. These sections were rinsed 3 times with PBS and incubated with DAPI (10 µg/mL) for 15 min. Then, they were examined under an inverted fluorescence microscope.
All of the data are presented as the mean ± SEM of at least three separate experiments, and statistical significance was evaluated using one-way ANOVA followed by Tukey's multiple comparison tests. Student’s unpaired t-test was used to compare two different groups. P < 0.05 was considered statistically significant.