Sunagawa, Take Matsuyama, Momo Fujii, Atsushi Kuwahara, Akiyoshi Kishino, Naoshi. Koide, Mototsugu Eiraku, Hidenobu Tanihara, Masayo Takahashi, and ...
Stem Cell Reports, Volume 10
Supplemental Information
Establishment of Immunodeficient Retinal Degeneration Model Mice and Functional Maturation of Human ESC-Derived Retinal Sheets after Transplantation Satoshi Iraha, Hung-Ya Tu, Suguru Yamasaki, Takahiro Kagawa, Motohito Goto, Riichi Takahashi, Takehito Watanabe, Sunao Sugita, Shigenobu Yonemura, Genshiro A. Sunagawa, Take Matsuyama, Momo Fujii, Atsushi Kuwahara, Akiyoshi Kishino, Naoshi Koide, Mototsugu Eiraku, Hidenobu Tanihara, Masayo Takahashi, and Michiko Mandai
Supplemental Experimental Procedures Animals NOG, NOG-rd1-2J, and NOG-rd10 mice used in these experiments were provided by Central Institute for Experimental Animals. Mice of type Rd1-2J (C57BL/6J-Pde6brd1-2J/J) and type rd10 (B6.CXB1-Pde6brd10/J) were obtained from the Jackson Laboratory by relocation. All mice were housed in 12-h cyclic light/dark with white light from fluorescent tubes of approximately 2.73 to 258.44 µW/cm 2 (about 10 to 825 lux). Mice with NOG background were isolated from other mice and provided with free access to food and distilled water. Development of NOG-rd1-2J and NOG-rd10 mice The first generation (N1) was obtained by in vitro fertilization (IVF) of male rd1-2J or rd10 mice and female NOG mice with embryo transfer (ET) to pseudopregnant MCH (ICR) (Figure 1). The resulting young female N1 mice were mated with male NOD mice until the N5 generation. Marker-assisted selection was performed on N5 mice to select the mice closest to the NOD strain, based on a previously reported protocol using 64 DNA micro satellite markers (Table 1) (Suemizu et al., 2008). The selected male mice and NOG females were used for IVF-ET to obtain both NOG-rd lines, which were confirmed by the genotyping procedures as described below. Genotyping To performed the marker-assisted selection of desired N5 mice, the IL2Rgnull mutations were genotyped using 2 sets of primers (Ito et al., 2002), and the SCID mutations were genotyped using Cycling Probe Technology (Higuchi et al., 2014). 64 microsatellite markers, one for each chromosome, were also analyzed to determine genetic backgrounds. PCR amplification of microsatellite loci was performed following the reported methods (Suemizu et al., 2008). Point mutations of Pde6brd1-2J and Pde6brd10 genes were identified by PCR amplification followed by the digestion with restriction enzymes NcoI-HF and NarI (BioLabs). The enzyme digestion with NcoI-HF results in 2 fragments of 146 bp and 98 bp in the mutant for rd1-2J genotyping, and the digestion with NarI results in 2 fragments of 293 bp and 297 bp in WT for rd10 genotyping. The primers are listed in Table S1. Flow cytometry The expression of CD4, CD8, CD19, NKp46, and CD11b on spleen cells from control rd mice (rd1-2J or rd10) and NOG-rd1-2J or NOG-rd10 mice was assessed by flow cytometry. After blocking mouse Fc receptors (1 x 106 cells were treated at 4 °C for 15 mins with anti-mouse FCRG3/FCGR2B antibodies from BD Pharmingen, Cat. No. 553141), the cells were incubated with primary antibodies or isotype control rat IgG (see Table S3) at 4 °C for 30 min. A secondary antibody (Alexa Fluor 488-conjugated anti-rat IgG, A-11006, Thermo Fisher Scientific) was used for cells with non-labeled primary incubation after wash at 4 °C for 30 min in the dark. All samples were examined with a BD FACSCanto™ II flow cytometer (BD Biosciences), and data were analyzed using the FlowJo 9.3.1 software package. Differentiation of hESC-retina Retinal differentiation by SFEBq, subconfluent hESCs were dissociated into single cells using TrypLe Select (Gibco) and seeded 12,000 cells per well of low-cell adhesion 96 well V bottomed plate (Sumitomo Bakelite) in differentiation medium (growth factor free CDM; gfCDM) containing DMEM/F12 Glutamax (Gibco), 10% KSR, 1% chemically defined lipid concentrate (Gibco), 450 uM monothioglycerol (Sigma-Aldrich), and supplemented with 10 uM Y-27632. At differentiate day (DD) 3, recombinant human BMP4 (final concentration = 1.5 nM; R&D systems) was added to each well. After DD 6, medium change half by half every third day. At DD15, aggregates were transferred to a low adhesion plate dish (Sumitomo bakelite) and cultured in DMEM/F12 Glutamax medium (Gibco) contained 1% N2 supplement (Gibco), 3 uM CHIR99021 (Wako), 5 uM SU5402 (Sigma-Aldrich) for 3 days. Aggregates were then cultured in NR-differentiation medium for long culture containing DMEM/F12 Glutamax medium (Gibco) and 1% N2 supplement (Gibco), 10% FBS (Gibco), 0.5 uM retinoic acid (Sigma-Aldrich), 0.1 mM taurine (Sigma-Aldrich), Penicillin-Streptomycin and Fungizone.
Differentiation of miPSC-retina Nrl-GFP transgenic miPSCs were generated from Nrl-EGFP mice (Akimoto et al., 2006; Homma et al., 2013). The genetically modified line Nrl-EGFP/ROSA::Nrl-CtBP2-tdTomato was differentiated into optic vesicle structures and cut into small pieces (around 0.5 mm × 2 mm) at differentiation day (DD) 13 for transplantation as previously described (Assawachananont et al., 2014; Mandai et al., 2017). Subretinal transplantation For graft preparation, transparent and continuous neural retinal epithelium part of hESC-retina and miPSCretina was cut into 0.5-mm-wide sections. Mice were anesthetized with isoflurane using an inhalation anesthetic system (Narcobit-E type II, Natsume Seisakusho, Tokyo, Japan). Anesthesia was maintained 2.0-5.0% isoflurane at an O2 flow rate of 1.5 L/min using a modified nosecone for mice. Pupils were dilated with MydrinP® (0.5% phenylephrine + 0.5% tropicamide; Santen Pharm Co., Osaka, Japan). The graft sheets were inserted to the subretinal space of 8-week-old NOG-rd1-2J as previously described (Assawachananont et al., 2014; Mandai et al., 2017). Electroretinogram (ERG) Mice were dark-adapted overnight and placed under dim red illumination (> 650 nm). Dark-adapted mice were anesthetized, followed by pupil dilation as aforementioned. Full-field ERG responses were sampled through a LED contact lens using a PuREC system (PC-100; Mayo Corporation, Aichi, Japan). Data were sampled at 1.98 Hz and band-pass filtered at 0.3-500 Hz. The stimulus intensity and duration were controlled using an electronic stimulator (LS-100, Mayo Corporation, Japan). An indifferent electrode was attached to the tongue and a ground electrode was clipped to the tail. For the scotopic ERG, the intensity of the light stimulation was increased from -3.0 to 2.0 log cd/m2/s, and the inter-stimulus interval was 10 s. In vivo ocular imaging Spectral-domain ocular coherence tomography (SD-OCT) images were obtained using the Envisu R2200 VHR SD-OCT (Bioptigen, Inc., Durham, NC, USA). Horizontal and vertical linear scans (1.4 mm in width, 1000 Ascans per B-scan and 100 B-scans per volume) were obtained. Funduscopy and fluorescent imaging were performed using the Micron IV camera (Phoenix Research Laboratories, Inc., Pleasanton, CA, USA). Fluorescein angiography imaging was performed using a Heidelberg retina angiograph (Heidelberg Engineering GmbH, Dossenheim, Germany) by injecting 0.1 mL fluorescein dye per 10 g body weight (1:15 dilution with physiological saline; Fluorescein Novartis 500mg/5mL, Novartis). Electron Microscopy Electron microscopy observation was processed as previously described (Assawachananont et al., 2014; Shirai et al., 2015). In brief, mice eyes were fixed with a mixture of 2% glutaraldehyde and 4% paraformaldehyde (PFA) in Phosphate Buffered Saline (PBS; pH7.4) overnight, corneas and lenses were removed, and 50-μm sections were made with a vibratome (PRO 7, DOSAKA EM Co., LTD., Japan). Retinal sections were stained with anti-RHO to confirm the presence of graft rosettes with mature ONL. Then, sections containing the graft were postfixed with ice-cold 1% OsO4 in 0.1M sodium cacodylate buffer, pH 7.3, for 2 h. Samples were then rinsed with distilled water, stained with 0.5% aqueous uranyl acetate for 2 h or overnight at room temperature, dehydrated with ethanol and propylene oxide, and embedded in Poly/Bed 812 (Polyscience). Ultrathin sections were cut, double-stained with uranyl acetate and Reynolds’ lead citrate, and viewed with a JEM1010 or JEM1400 transmission electron microscope (JEOL) at an accelerating voltage of 100 kV. Multi-electrode array (MEA) recording Animals transplanted with hESC-retina at post operation day 140 or more were dark-adapted for 1-3 days prior to MEA recording using the MED64 system (Alpha MED Scientific Inc) as previously described (Mandai et al., 2017). Alternatively, 12- to 13-week-old NOG-rd1-2J mice with or without miPSC-retina transplants (4 weeks post operation) were recorded using the USB-MEA60-Up-System (MultiChannel Systems, Germany) with the standard 8x8 probe (60MEA200/30iR-Ti-gr).
The animals were deeply anesthetized with sevoflurane or isoflurane inhalation, followed immediately by decerebration and harvest of retinas. After removal of vitreous body and identification of graft area, retinas were mounted on the electrodes with the ganglion cell side down and constantly perfused at 3-3.5 mL/min with warmed (35 ± 0.5°C), carbogenated (95% O2 and 5% CO2) Ames’ medium (A1420, Sigma-Aldrich). Opsinamide (10 µM; AA92593, Sigma-Aldrich) was added in the perfusion medium to suppress the melanopsin-driven RGC light responses during recording. Retinas were allowed to recover in the MEA chamber for 10-15 min before recording while monitoring the intrinsic hyperactive RGC firing as the feature of degenerated retina. Light responses to full-field white light stimuli in form of field potentials (microERG, or mERG) and RGC action potentials were recorded as previously described, followed by offline spike sorting and clustering analysis of RGC spikes. The full-field light stimuli were generated using a white LED source with irradiance ranging from 10-13 log photons/cm2/s at the focal plane of the electrodes, which approximately covered the low to high mesopic range. The regular light intensity used in this study (12.84 log photons/cm2/s) was the same as the intensity used in our previous works (0.45 log cd/m2/s). RGC responses to the same set of 3 repeated stimuli for each light intensity were recorded before and after 9-cis-Retinal (100 µM; R5754, SigmaAldrich) replenishment, as well as in the presence and absence of L-AP4 (20 µM; 016-22083, Wako) blockade to confirm the reproducibility. The procedures were conducted under dim red light with peak wavelength at 690 nm. The retinas were detached from the electrodes after recording for 1-2 h fixation with 4% PFA at room temperature for immunohistochemistry. RGC clustering after MEA analysis The MEA recording with either systems were sampled at 20 kHz online, then applied offline with the high pass filter at 1 Hz and 100 Hz for field potential and spike observation, respectively. For RGC clustering, the sets of 3 repeated recordings were spike sorted and then categorized by a homemade program based on deep learning system as previously described (Mandai et al., 2017). In brief, each RGC (spike source) was first grouped into light responsive or not (spontaneous) according to its spike patterns, and the light responsive ones were further clustered into 10 clusters including 3 ON patterns (transient, brisk-sustained, sustained), transient OFF, ONOFF and sustained ON-OFF, ON suppression, and 3 hyperactive patterns (ON, OFF, delayed). The clustering program requires an agreement of at least 2 of 3 repeated recordings on one of these patterns, or RGCs with inconsistent decision would be abandoned. Regarding Figure 5 and 6, normal response represents any of ON, OFF, and ON-OFF groups and hypersensitive response represents 3 hyperactive patterns. The results of clustering were also manually confirmed to remove the false-positive responses, if any. Immunohistochemistry For paraffin sections, eyes were fixed with SUPERFIX (Kurabo), embedded in paraffin (P3683; Sigma-Aldrich), and sectioned at 10-μm thickness using an automatic slide preparation system (AS-200; Kurabo). For cryosections, eyes were fixed in 4% PFA for 1 h at room temperature, cryoprotected in 30% sucrose in PBS overnight at 4 °C, embedded in OCT compound (Tissue Tec; Sakura Finetechnical), and sectioned at 10-μm thickness using a Leica CM3050S Research Cryostat (Leica Biosystems). Sections were blocked with 3% goat or donkey serum for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C, followed by incubation with corresponding secondary antibodies for 1 h at room temperature. The primary antibodies used were listed in Table S4. TUNEL assay was performed to detect the apoptosis of photoreceptor cells using the In Situ Cell Death Detection Kit, TMR red (12156792910, Roche Applied Science) according to the manufacturer's instructions. Synaptic 3D analyses using NOG-rd mice were performed as previously described (Shirai et al., 2015). In brief, eyes were fixed with 4% PFA and processed for cryostat sectioning at 50-μm thickness. The samples were preincubated with 5% goat serum and 3% Triton X-100 in PBS at 4 °C overnight, then incubated with primary and secondary antibodies at 4 °C for 2 days and overnight, respectively, before mounted with 2,20-thiodiethanol (Sigma-Aldrich). The whole-mount retinas after MEA recording were incubated with primary antibodies at 4 °C for 710 days, and subsequently incubated for 2 days with appropriate secondary antibodies. Samples were mounted using VECTASHIELD Antifade Mounting Medium (H-1000, Vector) and kept at 4 °C for long-term preservation.
Images of both sections and whole-mount preparations were acquired with a Leica-TCS SP8. 3dimensional (3D) imaging was performed as previously described with mild modification (Assawachananont et al., 2014; Mandai et al., 2017). Series of XY optical sections at system-optimized intervals was taken throughout the 50-µm section and reconstructed to obtain 3D sectional images using Imaris software (Bitplane). For the whole-mount retinal immunostaining, z-stack images were taken across the full sample depth and tiled automatically to cover the grafted and neighboring areas. Histological analysis Measuring the ONL thickness and counting the number of microglia in the ONL were performed in the retina at 0.5 mm to 1.5 mm from the optic nerve disc. Three retinal sections of one eye (n = 4-10 eyes for each time point) were included. We collected samples at the following time points: Rd1-2J mice; P7 (n = 5, 8 eyes), P14 (n = 5, 7 eyes), P21 (n = 5, 8 eyes), NOG-rd1-2J mice; P7 (n = 5, 7 eyes), P14 (n = 4, 6 eyes), P21 (n = 3, 6 eyes), P23 (n = 2, 2 eyes), Rd10 mice; P9 (n = 4, 6 eyes), P12 (n = 2, 4 eyes), P14 (n = 2, 4 eyes), P16 (n = 5, 8 eyes), P18 (n = 3, 6 eyes), P21 (n = 3, 6 eyes), P23 (n = 5, 8 eyes), P30 (n = 5, 8 eyes), NOG-rd10 mice; P9 (n = 3, 6 eyes), P12 (n = 3, 6 eyes), P14 (n = 3, 6 eyes), P16 (n = 5, 8 eyes), P23 (n = 5, 8 eyes), P30 (n = 4, 6 eyes), dark-reared rd10 mice; P23 (n = 2, 4 eyes), P30 (n = 3, 5 eyes), dark-reared NOG-rd10 mice; P23 (n = 3, 6 eyes), P30 (n = 2, 3 eyes). Modeling the ONL thickness We modeled the ONL thickness using a hierarchical Bayesian framework, and Markov Chain Monte Carlo (MCMC) sampling to estimate the model parameters. We performed this sampling using Stan 2.14.1 (Stan Development Team, 2016a), the RStan library 2.14.1 (Stan Development Team, 2016b), and R 3.3.2 (R Development Core Team, 2016). The ONL thickness of a certain strain of mouse, y µm, was modeled as a function of the rate of degeneration, a, the half-life period of ONL thickness degeneration, b days, the initial ONL thickness, c µm, and the observed time post birth, x days: 𝑦 =
𝑐 1 + 𝑎𝑒 𝑥−𝑏
(1)
To describe the variance in ONL thickness among individual mice, we modeled individual ONL thickness, ym µm, as a positive real parameter that was Gamma-distributed, with scale parameter θm: 𝑦 𝑦𝑚 ~𝐺𝑎𝑚𝑚𝑎 ( , 𝜃𝑚 ) 𝜃𝑚
(2)
Finally, the observed ONL thickness for each mouse, yobs, which is a positive real number, was also modeled as Gamma-distributed, with scale parameter θobs: 𝑦𝑚 𝑦𝑜𝑏𝑠 ~𝐺𝑎𝑚𝑚𝑎 ( ,𝜃 ) 𝜃𝑜𝑏𝑠 𝑜𝑏𝑠
(3)
Combining equations (1) through (3) with our experimentally obtained ONL thickness data, we estimated the posterior distribution of all parameters for each strain via MCMC sampling. The following priors were used for each parameter. Sampling was restricted to positive ranges.
a b c θm θobs
A Student-t distribution with the 4 degrees of freedom, mean 0, and variance 1. A uniform distribution from 0 to 100. A normal distribution with mean 60 and variance 10. A uniform distribution from 0 to infinity. A uniform distribution from 0 to infinity.
To test the potential of our model, we have simulated the ONL thickness over time by giving various values for a and b. The results showed that our model can describe a variety of degeneration patterns (or lack thereof) (Figure S2A). Figures S2B and S2C show the results of these analyses. Estimating the effect of strain and environmental light conditions to ONL thickness The effect of strain and environmental light conditions on the ONL thickness was estimated by fitting a model with a hierarchical Bayesian framework. For parameter estimation, the same method used in modeling the ONL thickness was applied. The ONL thickness of the ith mouse was modeled as a function of the strain and the light condition with the individual random effect. The explanatory variables were the categorical fixed effects fstrain, i and flight, i, and the random effect ri. With the parameters, ystrain, light can be modeled as: 𝑦𝑠𝑡𝑟𝑎𝑖𝑛,𝑙𝑖𝑔ℎ𝑡 = exp(𝑏𝑜 + 𝑏1 𝑓𝑠𝑡𝑟𝑎𝑖𝑛 + 𝑏2 𝑓𝑙𝑖𝑔ℎ𝑡 + 𝑟𝑖 ) 0, when Nog-rd10 mouse 𝑓𝑠𝑡𝑟𝑎𝑖𝑛 = { 1, when rd10 mouse 0, when cyclic light-reared 𝑓𝑙𝑖𝑔ℎ𝑡 = { 1, when dark-reared 𝑟𝑖 ~𝑁𝑜𝑟𝑚𝑎𝑙(0, 𝜎 2 )
(4)
The observed ONL thickness yobs was also modeled as Gamma-distributed, with the mean ystrain,light and scale parameter θobs: 𝑦𝑠𝑡𝑟𝑎𝑖𝑛,𝑙𝑖𝑔ℎ𝑡 𝑦𝑜𝑏𝑠 ~𝐺𝑎𝑚𝑚𝑎 ( , 𝜃𝑜𝑏𝑠 ) 𝜃𝑜𝑏𝑠
(5)
Combining equations (4) and (5) with our experimentally obtained ONL thickness data, we estimated the posterior distribution of all parameters at P23 and P30 via MCMC sampling. Uniform distribution was used for prior distribution for each parameter. Sampling was restricted to positive ranges for σ and θobs.
Supplemental References Akimoto, M., Cheng, H., Zhu, D., Brzezinski, J.A., Khanna, R., Filippova, E., Oh, E.C.T., Jing, Y., Linares, J.L., Brooks, M., et al. (2006). Targeting of GFP to newborn rods by Nrl promoter and temporal expression profiling of flow-sorted photoreceptors. Proc. Natl. Acad. Sci. 103, 3890–3895. Assawachananont, J., Mandai, M., Okamoto, S., Yamada, C., Eiraku, M., Yonemura, S., Sasai, Y., and Takahashi, M. (2014). Transplantation of embryonic and induced pluripotent stem cell-derived 3D retinal sheets into retinal degenerative mice. Stem Cell Reports 2, 662–674. Higuchi, Y., Kawai, K., Yamamoto, M., Kuronuma, M., Ando, Y., Katano, I., Nakamura, M., and Suemizu, H. (2014). Novel enhanced green fluorescent protein-expressing NOG mouse for analyzing the microenvironment of xenograft tissues. Exp. Anim. 63, 55–62. Homma, K., Okamoto, S., Mandai, M., Gotoh, N., Rajasimha, H.K., Chang, Y.-S., Chen, S., Li, W., Cogliati, T., Swaroop, A., et al. (2013). Developing rods transplanted into the degenerating retina of Crx-knockout mice exhibit neural activity similar to native photoreceptors. Stem Cells 31, 1149–1159.
Ito, M., Hiramatsu, H., Kobayashi, K., Suzue, K., Kawahata, M., Hioki, K., Ueyama, Y., Koyanagi, Y., Sugamura, K., Tsuji, K., et al. (2002). NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells. Blood 100, 3175–3182. Mandai, M., Fujii, M., Hashiguchi, T., Sunagawa, G.A., Ito, S., Sun, J., Kaneko, J., Sho, J., Yamada, C., and Takahashi, M. (2017). iPSC-derived retina transplants improve vision in rd1 end-stage retinal-degeneration mice. Stem Cell Reports 8, 1112–1113. R Development Core Team (2016). R: A Language and environment for statistical computing. R Found. Stat. Comput. http:// www.R-www.project.org/. Shirai, H., Mandai, M., Matsushita, K., Kuwahara, A., Yonemura, S., Nakano, T., Assawachananont, J., Kimura, T., Saito, K., Terasaki, H., et al. (2015). Transplantation of human embryonic stem cell-derived retinal tissue in two primate models of retinal degeneration. Proc. Natl. Acad. Sci. 113, E81–E90. Stan Development Team (2016a). Stan:AC++Library for Probability and Sampling. http://mc-org/. Stan Development Team (2016b). RStan: the R interface to Stan. http://mc-org/. Suemizu, H., Yagihashi, C., Mizushima, T., Ogura, T., Etoh, T., Kawai, K., and Ito, M. (2008). Establishing EGFP congenic mice in a NOD/Shi-scid IL2Rg(null) (NOG) genetic background using a marker-assisted selection protocol (MASP). Exp Anim 57, 471–477.
rd1-2J CD4+
T cells (helper T cells)
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100 50 80 1.4% 10.6% 10.6% 14.9% 40 60 60 30 40 40 20 20 10 20 0 0 1 2 3 4 0 0 1 2 3 4 0 10 10 10 10 10 10 10 10 10 10 100 101 102 103 104 PE-A PE-A PE-A CD4 CD4 CD4 60 CD8+ T cells 80 80 (cytotoxic T cells) 10.5% 13.0% 1.3% 60 40 60 20
0 0 1 2 3 4 0 0 1 2 3 4 0 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 APC-A APC-A APC-A CD8 CD8 CD8
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Figure S1. Characterization of immunodeficient state of NOG-rd1-2J and NOG-rd10 mice (related to Figure 1) FACS analysis of the spleen cells of 4 mouse lines. CD4+, CD8+, CD19+, and NKp46+ cell ratios were markedly lower in both NOG-rd1-2J and NOG-rd10 mice, in contrary to that in their counterparts, rd1-2J and rd10 mice, respectively.
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GFAP/GLUL/DAPI Figure S3. Apoptosis, infiltration or activation of immune cells and glial cells in the retina in NOG-rd1-2J and NOGrd10 (related to Figure 2) (A) AIF1 (Iba-1; green) and TUNEL (apoptotic nuclei marker; red) staining in rd10 and NOG-rd10 mice. (B) Comparison of the ONL thickness in cyclic light-reared (L/D; data from Figure 2C) and dark-reared (D/D) NOG-rd10 and rd10 mice at P23 and P30. The ONL thickness is drawn in the box-and-whisker plot, with the upper and lower whiskers extending to the largest or smallest value no further than 1.5 times of inter-quartile range from the box edges. To evaluate the light effect, the ONL thickness was mathematically modeled with the parameter of light condition. From the estimation, at P23, the possibility of rd10 (dark) > rd10 (cyclic light) was 99.2% and 99.9% in NOG-rd10 and rd10, respectively. At P30, the possibility of rd10 (dark) > rd10 (cyclic light) was 98.2% and 98.7% in NOG-rd10 and rd10, respectively. (C) Immune cells in the retina of 4 mouse lines at around the peak of inflammation (P14 for rd1-2J and NOG-rd1-2J, P23 for rd10, and P16 for NOG-rd10. Spleen cells as positive controls). Left to right: B cells, NK cells, neutrophils, cytotoxic T cells. (D) GFAP (glial fibrillary acidic protein, green) expression in 4 mouse lines co-stained with GLUL (glutamine synthetase; red). INL, inner nuclear layer; ONL, outer nuclear layer; also indicated by side bars for sectional views similarly in panel C, D. Scale bars, 20 μm (panel A, C), 50 μm (D).
A
B PRKCA
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Figure S4. Long term survival and maturation of transplanted human ESC-derived retinal sheets in NOG-rd1-2J mouse (related to Figure 4) (A-D) Immunohistochemistry of transplanted retina. PRKCA (PKCα; gray) (A) and CALB2 (Calretinin; gray) positive cells (B) were observed among the HuNu-positive graft cells. Some AIF1 (Iba-1) positive microglia (red) were observed both in the host retina and inside the transplants with ramified, non-active morphology (white arrows), and also inside the rosette (yellow arrow) next to isolated Crx::Venus positive cells (C-C’). (D) Müller glia were stained with GFAP (gray) and GLUL (glutamine synthetase; red). No gliotic barrier was observed between graft cells and host INL. (E-E”) A microglia-like cell was also observed inside the rosette by electron microscopy (yellow arrow). Magnified views show many oblique sectioned disc-like structures inside the rosette suggestive of outer segment formation by graft photoreceptors (orange arrowheads) (E’), and some of the disc fragments are engulfed by this microglia-like cell (orange arrows) (E”). Scale bars, 20 μm (panel A-D), 5 μm (E), 2 μm (E’) and 1 μm (E”).
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8-week NOG-rd1-2J central peripheral
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13W
ARR3 with DAPI
INL ONL
proximal retina mid-peripheral
distal retina central
peripheral
disc
Figure S5. Remaining cones and light-responsive activities in NOG-rd1-2J mice (related to Figures 5-7) (A) ARR3 (Cone Arrestin) is localized in the cone outer segments of C57BL/6 wild type retina as shown in the wholemount staining with the orthogonal view reconstructed from z-stack confocal images. (B) In 8-week-old NOG-rd1-2J, a few ARR3-positive cells were observed in the proximal retina (~1 mm from the optic nerve disc) and more in further peripheral retina (~2 mm from the disc) (n=4). (C) ARR3-positive cells were still found in 12-week-old NOG-rd1-2J retina but extremely few in the proximal area. (D) A few light responses were seen with MEA recording after recovery from L-AP4 in 12- and 13-week-old NOG-rd1-2J, but not in areas within 1-mm distance (indicated as pink shadow) from the disc (n=4). Normal and hypersensitive light responses were marked with red and blue arrows, respectively. ONL, outer nuclear layer. INL, inner nuclear layer. Scale bars, 20 μm (panel A-C).
Table S3. Antibodies used for flow cytometry, related to Figure S1 Antibody Name
Clone
Color
Vendor
Catalog Number
Anti-mouse CD4
H129.19
PE
BD Pharmingen
553652
Anti-mouse CD8a
53-6.7
APC
BioLegend
100711
Anti-mouse CD19
6D5
Non-labeled
BioLegend
115501
Anti-mouse CD335 (NKp46)
29A1.4
Non-labeled
BioLegend
137601
Anti-mouse CD11b
M1/70.15.11.5
APC
Miltenyi Biotec
130-091-241
Isotype control rat IgG2a, κ
RTK2758
Non-labeled
BioLegend
400501
Table S4. Antibodies used for immunohistochemistry, related to Figure 1-5 and S3-S5 Target Antibody (common name) (Protein Symbol)
Host Species
Dilution Vendor Factor
Catalog Number
RHO
anti-Opsin (RET-P1)
Mouse
1:1000
Sigma-Aldrich
O4886
OPN1MW
anti-Opsin, Red/Green (M/L cone opsin) Rabbit
1:1000
Merck Millipore
AB5405
OPN1SW
anti-Opsin, Blue (S cone opsin)
Rabbit
1:1000
Merck Millipore
AB5407
AIF1
anti-Iba-1
Rabbit
1:1000
Wako
019-19741
CD68
anti-mouse CD68
Rat
1:200
BioLegend
137001
CD19
anti-mouse CD19
Rat
1:100
BioLegend
115502
NCR1
anti-mouse CD335 (NKp46)
Rat
1:100
BioLegend
137601
LY6G
anti-mouse Ly-6G/Ly-6C
Rat
1:100
BioLegend
108402
CD8A
anti-CD8
Rabbit
1:100
abcam
ab203035
GLUL
anti-Glutamine Synthetase (GS-6)
Mouse
1:1000
Merck Millipore
MAB302
GFAP
anti-Glial Fibrillary Acidic Protein
Rabbit
1:500
Dako
Z0334-29-2J
PAX6
anti-Human Pax-6
Mouse
1:1000
BD Pharmingen
561462
VSX2
anti-Chx10
Sheep
1:1000
Exalpha
X1180P
RCVRN
anti-Recoverin
Rabbit
1:1000
Merck Millipore
AB5585
RARG
anti-RXRγ
Rabbit
1:500
Santa Cruz
sc-555
GFP
Anti-GFP
Rat
1:500
nacalai tesque
04404-84
POU4F1
Anti-Brn-3
Goat
1:200
Santa Cruz
sc-6026
ISL1
anti-Islet-1 homeobox
Mouse
1:500
DSHB
40.2D6
CALB1
anti-Calbindin D-28K
Rabbit
1:1000
Merck Millipore
AB1778
CALB2
anti-Calretinin
Rabbit
1:1000
Merck Millipore
AB5054
HuNu
anti-Nuclei (anti-Human Nuclei)
Mouse
1:500
Merck Millipore
MAB1281
PRPH2
anti-Peripherin 2
Rabbit
1:500
Proteintech
18109-1-AP
PRKCA
anti- Protein Kinase Cα
Rabbit
1:1000
Sigma-Aldrich
P4334
CTBP2
anti-CtBP2 (RIBEYE)
Mouse
1:500
BD Transduction Laboratories 612044
ARR3
anti-Cone Arrestin
Rabbit
1:500
Merck Millipore
AB15282
