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1 Cell Stem Cell, volume 10 Supplemental Information Self-Formation of Optic Cups and Storable Stratified Neural Retina from Human ESCs Tokushige Nakano, Satoshi Ando, Nozomu Takata, Masako Kawada, Keiko Muguruma, Kiyotoshi Sekiguchi, Koichi Saito, Shigenobu Yonemura, Mototsugu Eiraku, and Yoshiki Sasai Inventory of supplemental Information Figure S1: This figure is related to Figure 1. Figure S2: This figure is related to Figure 2. Figure S3: This figure is related to Figure 3 and Movie S3. Figure S4: This figure is related to Figure 4. Figure S5: This figure is related to Figure 5. Figure S6: This figure is related to Figure 6. Figure S7: This figure is related to Figure 7. Movie S1: This movie is related to Figure 2. Movie S2: This movie is related to Figure 2. Movie S3: This movie is related to Figure S3. Movie S4: This movie is related to Figure 4. Supplemental Experimental Procedures 1

2 Figure S1. Retinal differentiation culture and venus reporter cells. (A) Immunostaining of an optic cup self-formed from mouse ESCs (culture day 9; Eiraku et al, 2011) for Chx10 (red) and Pax6 (green). Dark blue, DAPI staining. (B) Cross section of the mouse embryonic optic cup at E10.5. DAPI staining. (C) Construction of the Rx::venus knock-in vector. (D) Southern blot confirming homologous recombination at Exon 1 of Rx gene. (E) Schematic of SFEBq culture with hescs. (F) Efficient reaggregation of hescs (time series; right three panels) using a V-bottomed 96-well plate with low-cell-adhesion coating (see the V 2

3 shape in the left-most panel). (G) Bright-field views of Figure 1C. (H) Mean values of Rx::venus-positive % in FACS analysis using 96 aggregates (three independent experiments). (I, J) Immunostaining for Sox2 (red; I) and N-cadherin (red; J; shown by bracket) in Rx::venus-negative portions (non-green) of the SFEBq aggregates on day 18. Nuclear staining with DAPI (I). Scale bars: 500 µm (G); 100 µm (A, B); 50 µm (I, J) 3

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5 Figure S2. Formation of optic vesicles from hescs (A) No significant difference in Chx10 expression between NR-selective and optic cup-forming (with CHIR99021) cultures (n =3 experiments; each experiment with 12 aggregates). (B) 3D reconstruction images of the day-17 optic vesicle. Multi-photon images. (C-F) Effects of other factors on the formation of Rx::venus + epithelia in hesc cluture. Fluorescence images of day-18 Rx::venus + epithelia (top row) and the corresponding bright-field views (bottom row) are shown. Unlike FBS (shown in Figure 1C), the addition of bovine fibronectin and vitronectin proteins (30 µg/ml, each; Life Laboratory)(D) to SAG-treated culture (control; C) did not have substantial Rx::venus-inducing effects. Conversely, the induction of Rx::venus + epithelia by 10% FBS was not inhibited when the culture was treated with the inhibitory peptides against fibronectin and vitronectin (100 µg/ml GRGDTP or 20 µg/ml Cyclic-RGDFV; Calbiochem)(E and not shown) or with FAK inhibitors (50 µm FAK inhibitor 14 or 1 µm PF573228; Tocris)(not shown). In contrast, dialyzed FBS (Gibco) had a similar Rx::venus-inducing effect (F) as seen with FBS, while the addition of IGF (200 ng/ml), Fgf2 (100 ng/ml) or Activin (50 ng/ml) showed no substantial effects (not shown). Error bars show s.e.m. Scale bars: 500 µm (C, D, E, F) ; 200 µm (B) 5

6 Figure S3. Self-organized formation of optic cups from hescs (A) Live-imaging of self-formation of an Rx::venus + optic cup. Invagination of the distal epithelium in the hesc-derived optic vesicle is shown. (B) 3D reconstruction view (half cut) of the day-26 optic cup self-formed in 3D hesc culture (Rx::venus + ). (C) Day-26 hesc-derived optic cup accumulating pigments (arrowheads) in the proximal RPE portion, where differentiation is more advanced. Scale bars: 100 µm (A); 50 µm (B, C) 6

7 Figure S4. Self-driven eversion of hescs-derived NR epithelia (A) Schematic of the NR excision from the optic cup after invagination. The direction of bending (apically convex) has been already fixed at this stage in both mesc and hesc cultures, and does not change after excision. 7

8 (B-G) Immunostaining of day-22 NR epithelia (carrying Rx::venus ; excised on day 18) for phospho-paxillin (ppaxillin; red; B,C), Ki67 (red; D), phospho-histone H3 (ph3; light blue; D), active Caspase 3 (ActCasp3; red; E) and Phalloidin (F). Dark blue, DAPI. Right, treatment with anti-integrin antibody; left, control treatment. The anti-integrin-ß1 antibody inhibited the continuously high ppaxillin accumulation on the basal side (C, left), facing to the basement membrane. Low ppaxillin accumulation was seen discontinuously as discrete dots (C, right). No clear effects on Ki67, ph3, apoptosis (ActCasp3) or actin accumulation (Phalloidin). Scale bars: 100 µm (B, D, E, F); 20 µm (C). 8

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10 Figure S5. Formation of an apical photoreceptor layer in hesc-derived NR epithelium (A) Vector construction for the homologous recombination for Crx::venus-knock-in. (B-D) Crx + photoreceptor precursors located in the apical-most and intermediate-deep zones on day 42. Blimp1 expression was mostly found in Crx::venus + cells (C). Brn3 + ganglion cells were located on the basal zone (D), while the Tuj1 + axonal bundles were seen on the basal surface (arrowheads). (E) Coexpression of Crx::venus and Recoverin in the day-60 NR. (F,G) Crx::venus + cells (day 60) did not coexpress the proliferation maker Ki67 (F) or Brn3 (G), while many Brn3 + cells coexpressed Tuj1 (G). (H-M) Immunostaining of day-126 NR epithelia. (H, I) Accumulation of M/L-opsin (M-Opsin) in the apical-most portion of the photoreceptor zone. (J) Brn3 + cells in the Tuj1 + basal zone. As previously described for late retinal development in vivo (Galli-Resta and Ensini, 1996), the Brn3 + cell percentage gradually decreased in vitro as the culture was extended (particularly during day ). (N-P) Transmission electron microscopic analysis of the apical surface of day-126 NR epithelia. Protruding and mitochondria-rich structures similar to the ellipsoids of photoreceptor inner segments were seen, and had apical cilia reminiscent of the connecting cilia of photoreceptors. Some apical cilia had a membrane vesicle on their tip (P, arrow). Scale bars: 100 µm (B, D, E, F); 50 µm (C, G, J); 20 µm (H, I, K, L, M); 1 µm (N, O, P) 10

11 Figure S6. Notch inhibition promoted photoreceptor differentiation in hesc-derived NR epithelium (A-C) Control NR epithelia (excised on day 18) in isolation suspension culture. Generation of Crx::venus + 11

12 photoreceptor precursors was only gradual during days (D-F) NR epithelia treated with the Notch inhibitor DAPT from day 29. A moderate increase in Crx::venus + photoreceptor precursors was seen after 4 days of DAPT treatment (E). After 12 days of DAPT treatment, a dramatic induction of Crx::venus + photoreceptor precursor was observed (F). (G-J) Isolated NR culture treated with DMSO only (G,I) or with DAPT (H,J) during days On day 33, four-day treatment with DAPT already exhibited substantial inducing-effects on Crx::venus + photoreceptor generation. The increase was particularly obvious in the apical zone (top side in H). There, Crx::venus + photoreceptor cells appeared as clusters that extended basally. Scale bars: 200 µm (A, B, C, D, E, F); 100 µm (G, H); 25 µm (I, J). 12

13 Figure S7. Efficient cryopreservation of hesc-derived NR epithelia (A) Average fluorescent density of Rx::venus in NR epithelia (15 NR epithelia/condition) five days after thawing. The DMSO+ethylene glycol+sucrose conditions yielded the best cryopreservation efficacy for the maintenance of Rx::venus expression. (B,C) In contrast to vitrification with pretreatment in DMSO+ethylene glycol+sucrose, the conventional slow freezing with DMSO did not maintain Rx::venus expression (C, bottom). (D) Day-126 NR epithelia (carrying Rx::venus) frozen on day 105 and fixed on day 126. Immunostaining for Calretinin. (E) NR 13

14 epithelium (Crx::venus) frozen on day 33, thawed, and then treated with the vehicle DMSO (left panels) or DAPT (right panels) during days Crx::venus expression was dramatically augmented by DAPT treatment. (The DMSO-treated control also expressed Crx::venus in a speckled manner; only dim Crx::venus signals of the controls were seen in these panels, because the exposure of camera was adjusted to the very bright signals of the DAPT-treated ones.) Scale bars: 500 µm (B, C, E); 50µm (D). 14

15 Supplementary Movies Movie S1 Evagination of Rx::venus + vesicle in hesc culture (days 14-17) Rx::venus hescs cultured under the optic-cup-forming conditions. Movie S2 Interkinetic nuclear migration in hesc-derived NR (day 20; 35-hour recording) Visualized by partial mixture of Rx::venus hescs at initial reaggregation. Movie S3 Invagination of hesc-derived NR (days 20-23) Rx::venus hescs cultured under the optic-cup-forming conditions. Movie S4 Self-driven eversion of hesc-derived NR (day 18-21) Thickened epithelium was excised manually from hesc-derived retinal epithelium on day 18, and further cultured en bloc. 15

16 Supplemental Experimental Procedures Culture of hescs hescs (KhES-1) were used following the hesc research guidelines of the Japanese government. hescs were maintained on a feeder layer of MEFs inactivated by mitomycin C treatment in DMEM/F12 (Sigma) supplemented with 20% (vol/vol) Knockout Serum Replacement (KSR, Invitrogen), 2 mm glutamine, 0.1 mm nonessential amino acids (Invitrogen), 5 ng/ml recombinant human bfgf (Wako), 0.1 mm 2-mercaptoethanol (2-ME), 50 U/ml penicillin and 50 µg/ml streptomycin under 2% CO 2. For passaging, hesc colonies were detached and recovered en bloc from the feeder layer by treating them with 0.25% trypsin and 0.1 mg/ml collagenase IV in PBS containing 20% KSR and 1 mm CaCl 2 at 37 C for 7 min. The detached hesc clumps were broken into smaller pieces (several tens of cells) by gentle pipetting. The passages were performed at a 1:3 to 1:4 split ratio. For SFEBq culture, hescs were dissociated as described in Experimental Procedures and cultured with Y-27632, which inhibits dissociation-induced apoptosis. The Y concentration (initially 20 µm) was reduced in a step-wise manner by replacing half the medium with medium lacking Y-27632, as in Figure 1A. After culturing in a V-bottomed 96-well plates for 12 days, the aggregates were transferred to a petri dish for suspension culture in retinal differentiation medium (96 aggregates per 10-cem dish). Optic cup size The size of the optic cup was assessed by measuring the long-axis diameters (surface-to-surface) of 8 samples using the BZ-9000 system and BZ-II analyzer software (Keyence). Regarding the difference in the optic cup size, it does not seem to be critical that SFEBq culture conditions for hescs and mescs are different in the initial cell numbers per aggregate (9000 and 3000, respectively; empirically, hescs form well-growing spheres more efficiently at 9000 cells; Watanabe at al, 2007; Eiraku et al, 2008), since mescs did not form a larger optic cup even when the culture was started with 9000 cells (our unpublished observations). Notch inhibition and teratoma assay The Notch inhibition experiment was performed by treating excised NR tissue with 10 µm DAPT during the period indicated in the text. The teratoma experiments were performed by injecting hesc-derived NR tissues (~10 6 cell-equivalent) into SCID mouse testes. No teratoma formed (n = 12; 9 weeks). In contrast, 5x10 4 undifferentiated hescs were sufficient to form teratoma reproducibly. Generation of knock-in hesc lines Our gene-targeting strategy is illustrated in Fig. S1C and Fig. S5A. A site-specific zinc-finger nuclease (ZFN; Porteus, 2008) was used to facilitate homologous recombination in hes cells. The ZFN designed to cause a double-strand break in exon1 of human Rx/RAX gene was purchased from Sigma-Aldrich (St. Louis, MO). To 16

17 generate the targeting construct, the 5 arm (2.0 kbp) and 3 arm (1.0 kbp) were amplified by PCR from KhES-1 genomic DNA. The cdna of venus, encoding a yellow variant of GFP, was fused in-frame into the first exon of Rx/RAX gene at the initial ATG. A PGK promoter-driven neomycin-resistance selection cassette flanked by loxp sites was inserted downstream of venus. For transfection of the targeting vector and ZFN-coding mrnas, hescs were dissociated into single cells in TrypLE Express (Invitrogen) containing 0.05 mg/ml DNase I (Roche) and 10 µm Y-27632, and cells were resuspended in NEON Resuspension Buffer R and electroporated using the Neon transfection system (Invitrogen) with protocol No.17 (850V, 30 milliseconds, 2 pulse). Transfected cells were plated onto Neo-resistant feeder MEF cells. Homologous-recombinant hescs selected with 50 µg/ml Geneticin (Gibco) were screened by PCR genotyping and further confirmed by Southern blot with the 5 and neo probes (Figure S1D). The floxed PGK-neo cassette was removed from clone RxVA22 (this enhanced the expression of Venus) by transient transfection with the Cre-expressing plasmid (pcag-gs-cre) using FuGene HD (Roche) and the resultant subclone RxVA22-N37 was established. RxVA22-N37 was mainly used for the experiments shown in this report, although indistinguishable results were obtained using other sublines. hescs with venus knocked in at the CRX locus were also generated by a similar method as described above (Figure S5A). Immunohistochemistry, qpcr and FACS Immunohistochemistry was performed as described (Eiraku et al, 2011) with primary antibodies described below. The antibodies were used at the following dilutions: GFP (rabbit/1:500/mbl), (rat/monoclonal/1:500/nacalai Tesque) or (mouse/monoclonal/1:1000/invitrogen), Pax6 (rabbit/1:200/covance), Chx10 (goat/1:100/santa Cruz) or (sheep/1:500/exalpha), Mitf (mouse/monoclonal/1:500/exalpha), NRL (goat/2 µg/ml/r&d), apkc (rabbit/1:100/santa Cruz), Laminin (mouse/monoclonal/bd/1:500), Tuj1 (mouse/monoclonal/covance/1:500), Blimp1 (rat/monoclonal/1:100/santa Cruz), Brn3 (goat/1:100/santa Cruz), Ki67 (mouse/monoclonal/1:200/bd), Recoverin (rabbit/1:500/chemicon), RXR-gamma (rabbit/1:2.5/spring Bioscience), ZO-1 (rabbit/1:200/invitrogen), Ptf1a (Armenian Hamster/monoclonal/1:2; kind gift from Dr. Yuichi Ono, KAN Institute), Rhodopsin (mouse/monoclonal/1:1000/sigma; RET-P1), S-opsin (rabbit/1:500/millipore), M-opsin (goat/1:100/sanata Cruz), Sox2 (goat/1:250/santa cruz), pmlc2-ser19 (Rabbit/1:500/CST), Acetyl-Tubulin (Mouse/1:200/Abcam), N-cadherin (Mouse/1:500/BD), Active Caspase3 (Rabbit/1:500/CST), Phospho-Paxillin (Rabbit/1:500/CST), GABA (Mouse/1:500/Sigma), Calbindin (Rabbit/1:500/Millipore), Calretinin (Rabbit/1:1000/Chemicon) and phospho-histone H3 (rabbit/1:500/upstate). Counter nuclear staining was performed with DAPI (Molecular Probes). Phalloidin staining was performed with Alexa Fluor 647 phalloidin according to the manufacturer's instructions. Immunostaining with Ptf1a antibody required an antigen retrieval step in 1x HistoVT One solution (ph 7.0, 20 min at 70 C; Nacalai Tesque). Quantitative PCR (12 aggregates/sample) was performed using a 7900 HT Fast Real Time PCR System (Applied Biosystems) and data were normalized to Rx/RAX expression. TaqMan gene expression assays were used: 17

18 Hs _s1 (CHX10), Hs _m1 (MITF) and Hs _m1 (Rx/RAX). For FACS analysis, cells (from 96 aggregates per experiment) were dissociated to single cells by Accumax (Innovative Cell Technologies) treatment, analyzed at 4 C and counted with FACSAria (Becton Dickinson). The data were analyzed with FACSDiva software (Becton Dickinson). Electron Microscopy For electron microscopy, hesc aggregates were fixed in 2.5% glutaraldehyde and 2% formaldehyde in 0.1 M cacodylate buffer (ph 7.2) and then processed for thin sectioning and transmission electron microscopy. Live imaging of optic-vesicle invagination 3D live imaging was performed as described (Eiraku et al, 2011) using specially assembled inverted microscopes (confocal or multi-photon) combined with a full-sized CO 2 /O 2 incubator. The position of the ESC aggregate or isolated vesicle was fixed in a drop of undiluted Matrigel, which was then immersed in culture medium (described above) on a 3.5-cm glass-bottom dish (Movies S1 and S2), or by placing it in a small silicon rubber insert on the glass bottom dish (Culture Insert; ibidi) (Movies S3 and S4). For confocal analysis, optical section images were obtained using a 20x objective lens (Olympus), a spinning disk confocal system (CSU-X1, Yokogawa) and an EMCCD camera (Andor, 512x512 pixels). The incubation system is based on LCV-110 (Olympus), but the optical system including bilateral telecentric lenses was newly designed (Eiraku et al, 2011). To obtain the whole aggregate view, a 0.5x lens was interposed in the light path. Live imaging of NR eversion was performed with the LCV-CSU-X1 system without embedding in gel. For high-resolution multi-photon live imaging, a stack of optical section images (512x512 pixels for the X Y plane and 2 µm for Z-axis step; typically 150 sections) was captured at each time point using a 25x water-immersion lens (N.A. 1.05, Olympus) and multi-photon femtosecond laser (900nm; Mai-Tai DeepSee ehp, Spectra-Physics) with group velocity dispersion auto-compensation. The 3D image analyses were done with Imaris 7.1 (Bitplane). The bright-field view in parallel to multiphoton imaging was obtained by scanning with a 900-nm femtosecond laser and detecting the transmission light with a separate photo-multiplier. Reference for Supplemental Information Galli-Resta, L. and Ensini, M. (1996) An Intrinsic Time Limit between Genesis and Death of Individual Neurons in the Developing Retinal Ganglion Cell Layer. J. Neurosci. 16, Porteus M. (2008) Design and testing of zinc finger nucleases for use in mammalian cells. Methods Mol Biol. 435,