All animals were treated in accordance with the guidelines defined by the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmology and Vision Research, and were bred and managed according to the Animal Experiment guidelines of the Asan Medical Center. Our experimental methods were reviewed and approved by the Institutional Animal Care and Use Committee (approval number 2017-12-169).
Pde6b knockout rats and SD rats (OrientBio, Gyeonggi, Korea) were housed in a specific-pathogen-free environment on a 12 h/12 h light–dark cycle at the Asan Medical Center. In the present study, hiPSC-derived retinal cells were transplanted into Pde6b knockout rats at the age of 2–3 weeks. hiPSC-derived retinal cells were subretinally injected into the right eye, whereas vehicle (phosphate-buffered saline; PBS) was injected into the left eye as an untreated control. The rats were euthanized 2 weeks, 2 months, or 4 months after transplantation, and the eyeballs were removed for analysis.
Differentiation of hiPSCs into retinal cells
To induce differentiation of hiPSCs into retinal cells, hiPSCs were plated at a density of 2 × 104/cm2 in vitronectin-coated 12-well plates and cultured until 80% confluency was achieved. Then, the cells were cultured for 14 days in neurobasal induction medium; the induction medium was replaced every day to initiate RPE and photoreceptor cell differentiation. The neurobasal induction medium consisted of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with F12, 1× N2, 1× B27, and 1× nonessential amino acids (Life Technologies), and the following cytokines at different stages of the differentiation process: differentiation day 0–1: 10 mM nicotinamide (Sigma), 50 ng/ml noggin, 10 ng/ml DKK-1, and 10 ng/ml insulin-like growth factor-1 (IGF-1; Peprotech); differentiation days 2–3: 10 mM nicotinamide, 10 ng/ml noggin, 10 ng/ml DKK-1, 10 ng/ml IGF-1, and 5 ng/ml FGF2; differentiation days 4–5: 10 ng/ml DKK-1, 10 ng/ml IGF-1, and 100 ng/ml activin A (Peprotech); differentiation days 6–7: 100 ng/ml activin A, and 10 µM SU5402 (Sigma); and differentiation days 8–14: 100 ng/ml activin A, 10 µM SU5402, and 3 µM CHIR99021 (Peprotech). Fasudil (10 µM, Adooq), a ROCK inhibitor, was added to the medium on all differentiation days19.
At the end of the 14-day RPE and photoreceptor cell differentiation process, the cells had reached a density of ~1 × 105 cells/cm2 in vitronectin-coated dishes. On day 14, the cells were passaged using Accutase (Gibco) and cultured in X-Vivo medium (Lonza) supplemented with 10 µM fasudil to support the enrichment, and expansion of the RPE and photoreceptor cells. The medium was replaced every three days after each subculture step.
Characterization of hiPSC-derived retinal cells by immunocytochemistry
Differentiated cells were fixed with 4% formaldehyde overnight at 4 °C and washed twice with phosphate-buffered saline (PBS). The cells were permeabilized and blocked through incubation in blocking solution comprising 1% BSA, 0.1% Triton X-100, and 0.1% Tween 20 in PBS for 30 min at room temperature. After this, the cells were incubated overnight at 4 °C with primary antibodies against melanocyte-inducing transcription factor (MITF; Abcam, 1:200) and CHX10 (Santa Cruz, 1:200), which are RPE cell and neural retina markers, respectively. The primary antibodies were then removed by washing with 0.1% Tween 20 in PBS, and the cells were incubated at room temperature for 1.5 h with the following secondary antibodies: goat anti-rabbit IgG H&L (1:500, Alexa Fluor 555-conjugated, Abcam) for MITF and goat anti-mouse IgG H&L (1:1000, Alexa Fluor 488-conjugated, Abcam) for CHX10. After antibody incubation was completed, the nuclei were counterstained by incubation with 100 ng/ml 4′,6-diamidino-2-phenylindole (DAPI, Sigma) in PBS for 30 min at room temperature. All fluorescence images were acquired using a fluorescence microscope (AxioObserver Z1; Carl Zeiss, Oberkochen, Germany).
A sclerotomy was initially made using a sterile 26-gauge needle (Video 1). Then, 5 µl of an hiPSC-derived retinal cell suspension with a concentration of 7.4 × 105 cells/μl was injected into the subretinal space of the right eyes of Pde6b knockout rats, using a Hamilton syringe with a 33-gauge needle through the previously made sclerotomy. PBS served as a control and was injected into the left eye. The cells were washed with PBS before transplantation and dissociated to allow the release of individual cells into X-Vivo medium. Pde6b knockout rats (aged 2–3 weeks) were anesthetized by intraperitoneal administration of a mixture of 0.4 ml/kg xylazine hydrochloride, 0.6 ml/kg tiletamine hydrochloride, and zolazepam hydrochloride. The pupils were dilated using tropicamide and phenylephrine hydrochloride eye drops (Mydrin-P; Santen Pharmaceutical Co., Ltd., Osaka, Japan). Subretinal injection was performed under a surgical microscope (Carl Zeiss). After conjunctival peritomy, a 26-gauge needle was used to inject the cells. Then, using a Hamilton syringe with a 33-gauge needle, 5 μl hiPSC-derived retinal cells were injected into the subretinal space. After the injection, hypromellose (Hycell solution 2%; Samil Pharmaceutical Co., Ltd., Seoul, Korea) was applied to prevent drying of the eye, and an infrared lamp was used to maintain body temperature.
Retinal cross-sectional images were obtained 2 weeks, 1 month, 2 months, 5 months, and 10 months after transplantation, using optical coherence tomography (OCT; IIS Science, Korea). Using B-scan OCT angiography, we obtained angiographic images of the retinal layers and retinal surfaces. These images were obtained using the same protocols used for anesthesia administration and pupillary dilation. During OCT imaging, hypromellose was applied to both eyes to prevent excessive drying of the ocular surface during recovery. The animals were placed on a heating pad, and the eye of interest was positioned directly in front of the scan head lens.
The rats were housed in the dark for >12 h before electroretinogram (ERG) recording was performed, and only dim red light was used for preparation in a darkroom. Anesthesia administration and pupillary dilation were performed using the same protocols as those used in the transplantation procedure. For OCT imaging, hypromellose was topically applied to prevent drying of the eyes. The rats were placed on a heating pad to maintain body temperature. Two electrode needle tips were inserted between the eyes, and another was inserted under the tail skin. A gold lens was then placed near the cornea to measure the electrical signals from the retina. A Granzfeld stimulator was used to deliver a total of three to ten green flashes, and the results were averaged for analysis. Flashes of 1.3 log cd/m2 green light were used for scotopic ERG with a sampling rate of 1000 Hz and bandpass filter of 2.5 mV. After recording, infrared light was used to maintain body temperature until the animals regained consciousness.
Human mtDNA validation using conventional PCR and Sanger sequencing
Retinal tissue samples containing hiPSC-derived retinal cells were harvested 2 weeks and 4 months after transplantation, and paraffin-embedded sections (10 µm thick) were observed under an Olympus CX41 microscope (Olympus America, Center Valley, PA, USA). Mitochondrial DNA (mtDNA) was performed using a PicoPure® DNA Extraction Kit (Applied Biosystems). PCR master mix (AccuPower® PCR Master Mix) and primers (F-5′-GCCTTCCCCCGTAAATGATA-3′ and R-5′-CTTCTGTGGAACGAGGGTTT-3′; mtDNA 15 S rRNA) were used to detect transplanted mtDNA of human origin. The final PCR volume was 20 µl, which included 10 µl PCR master mix, 1 µl DNA template, 7 µl nuclease-free water, and 1 µl each of the forward and reverse primers. The DNA template concentrations were 500 and 5000 ng/µl for the samples harvested 2 weeks and 4 months after transplantation, respectively. The positive and negative controls were hiPSC mtDNA and mtDNA obtained from the retinas of Pde6b knockout rats, respectively. PCR was performed under the following thermal cycling conditions: initial denaturation at 95 °C for 1 min; 35 cycles of 95 °C for 15 s, 56 °C for 15 s, and 68 °C for 1 min; and a final elongation at 68 °C for 3 min. The amplified samples were then electrophoresed at 100 V on a 2% agarose gel and visualized using a GelDoc XR+ Imaging System (Bio-Rad).
Hematoxylin and eosin staining
Two weeks, 2 months, and 4 months after transplantation, the animals were euthanized using a CO2 chamber, and the eyeballs were harvested. Enucleated eyes were fixed in Davison’s buffer for 6 h and embedded in 4% formalin solution overnight. Vertical sections with a thickness of 4 µm were prepared, mounted on slides, stained with hematoxylin and eosin (H&E), and viewed under an Olympus CX41 microscope (Olympus America, Center Valley, PA, USA).
Immunohistochemical analysis of retinal tissue
Deparaffinization was performed by washing the slides twice with 100% ethanol; then, the slides were rehydrated by washing with 95, 80, and 75% ethanol. The slides were then placed in 1× antigen retrieval solution (10× citrate buffer, ImmunoBioscience, #AR-6544-05) and boiled in a pressure cooker (Instant Pot Duo Multicooker; Instant Brands Inc., Canada) for 30 min at 120 °C and 10 min at 90 °C. After cooling the slides to room temperature, blocking was performed using 5% goat serum (normal goat serum, S-1000; Vector Laboratories) and 0.1% Triton in PBS. The slides were incubated with the appropriate primary antibodies overnight at 4 °C, and subsequently with secondary antibody for 1 h at room temperature. The slides were mounted using mounting solution with DAPI (VECTASHIELD® Hardset Antifade Mounting Medium, H-1400; Vector Laboratories).
Human TRA-1-85/CD147 (MAB3195; Millipore, 1:100 dilution), MITF (AB122982; Abcam, 1:100 dilution), Recoverin (AB5585; Millipore, 1:100 dilution), PDE6B (sc-377486; Santa Cruz Biotechnology, 1:100 dilution), and CD45 (AB10558; Abcam, 1:100 dilution) primary antibodies were used to label human cells, RPE cells, photoreceptor cells, and inflammatory cells, respectively. DyLight 594-conjugated (DI-1594; Vector Laboratories, 1:1000) and FITC-conjugated (A-11029; Invitrogen, 1:1000) secondary antibodies were used. Images were taken using a Zeiss LSM 880 upright confocal microscope (Carl Zeiss, Germany). DAPI staining was used to identify and count cells in the retinal area of interest. Costaining with TRA-1-85/CD147 was performed to identify cells of human origin20.
Statistical analysis was performed using GraphPad Prism 7.0. Normal distribution was verified using the Shapiro–Wilk test. Statistical data are presented as the mean ± standard error of the mean, and p values were determined using the Mann–Whitney U test and unpaired two-tailed Student’s t test.