Supporting InformationMeyer et al. 10.1073/pnas.0905245106SI TextMaintenance of hESCs and Human iPS Cells. Human embryonic stemcells (H9 line) and induced pluripotent cells (IMR90 clone 4)were expanded on a feeder layer of irradiated mouse embryonicfibroblasts (MEFs) in embryonic stem cell medium (ESCM)consisting of DMEM/F12 (1:1), 20% knockout serum replace-ment, 0.1 mM �-mercaptoethanol, 1 mM L-glutamine, MEMnonessential amino acids, and 4 ng/mL FGF2. Cells were pas-saged every 5–6 days, and morphologically identifiable differ-entiated cells were mechanically removed at each passage.

Differentiation of hESCs and Human iPS Cells. The procedure fordifferentiating hESCs and human iPS cells toward an eye fieldfate was performed with modifications to a previously describedprotocol (1). hESCs and iPS cells were enzymatically lifted fromthe MEF cells with dispase (1 mg/mL) and grown as aggregatesin suspension in ESCM without FGF2 for 4 days to initiatedifferentiation. The aggregates were then switched to a chemi-cally defined neural induction medium (NIM), which consistedof DMEM/F12, 1% N2 supplement, MEM nonessential aminoacids, and 2 �g/mL heparin. Two days later, aggregates wereallowed to attach to the surface of the culture dish with theaddition of laminin. Within a few days, columnar cells developedand formed neural tube-like structures. To allow for retinaldifferentiation, the medium for the hESC- and iPS cell-derivedneuroepithelial rosettes was switched to a chemically-definedretinal differentiation medium (2) consisting of DMEM/F12(3:1) supplemented with 2% B27 on day 16 of differentiation.For neural retinal progenitors, rosettes were mechanically iso-lated with light trituration from adherent cultures upon changeof medium at day 16, effectively keeping the rosettes intact whileleaving behind the most peripheral cells of the colonies. Theseclusters were then placed in suspension culture in non-adherentculture dishes to allow for neurosphere formation. For RPEdifferentiation, neuroepithelial rosette-containing colonies werekept attached to the culture surface in RDM. In separateexperiments, BMP4 and Wnt3A (R&D Systems; 100 ng/mL eachfrom day 0 to day 10) or SU5402 (Calbiochem; 10 �M from day0 to 10 or day 16 to 40) was added to differentiating hESCcultures.

RT-PCR. Total RNA was isolated from cell cultures from variousstages of differentiation using the RNAeasy kit (Qiagen) andtreated with DNase I. Reverse transcription was performed withthe SuperScript III RT-PCR kit (Invitrogen). PCR was per-formed with GoTaq PCR master mix (Promega) and subsequentPCR products were run on 2% agarose gels. For quantitativeRT-PCR (qPCR), reactions were performed with Sybr GreenSupermix (Applied Biosystems) and the Opticon 2 DNA Engineand Opticon Monitor 2.02 software (MJ Research). Primer setsused are listed in Table S1. All PCR reactions were run at 30cycles unless otherwise indicated, and all qPCR reactions wererun at 40 cycles.

Immunocytochemistry. hESC and iPS cell aggregates or neuro-spheres were plated onto polyornithine- and laminin-coatedcoverslips overnight to allow for attachment, and then fixed with4% paraformaldehyde. Cells were then permeabilized in 0.2%Triton X-100 for 10 min. For Mitf immunocytochemistry, cellswere incubated in ice-cold 90% methanol for 10 min as anadditional permeabilization step. Immunostaining was per-formed in 0.1% Triton X-100 and 5% donkey serum using theantibodies listed in Table S2. Labeled cells were visualized witheither Alexafluor 488- or Cy3-conjugated secondary antibodies,and nuclei were counterstained with either Hoechst or To-Pro-3nuclear dyes. Images were obtained from a Nikon TE600fluorescent microscope equipped with a SPOT camera andsoftware or from z-stacks of cell clusters obtained on a Nikon C1Laser Scanning Confocal microscope.

FACS. Cells were dissociated with either trypsin or Accutase(Chemicon), washed with a fluorescence-activated cell sorting(FACS) buffer (PBS, 0.1% sodium azide, and 2% donkeyserum), and fixed with 0.1% paraformaldehyde for 10 min. Cellswere then permeabilized with ice-cold 90% methanol for 20 minand incubated overnight in primary antibodies at a concentrationof 1 �g of antibody per 1 million cells in FACS buffer. Immu-nostaining was then completed with either donkey-anti-mouseor donkey-anti-rabbit Alexa 488 secondary antibodies for 2 h,after which cells were washed with FACS buffer, and then sortedwith a Becton Dickinson FACSCaliber. A nonspecific, species-appropriate IgG negative control was included in all FACSexperiments. Data retrieved from the sorting was analyzed withCellQuest Pro software (Becton Dickinson).

Quantification of Immunocytochemistry Data. Counts were obtainedfrom immunolabeled cell clusters plated on coverslips. Thenumber of cells expressing a particular marker was counted andcompared to the total number of cells found in each field,typically as determined by Hoechst 33358 or ToPro-3 nuclearstaining. For all quantifications, a minimum of four coverslipswere stained from each of at least three separate experiments.Results were expressed as mean � standard error.

Western Analysis. Western blots were performed as previouslydescribed (3). Briefly, 20 �g protein samples obtained from celllysates were separated on 4% to 20% gradient Tris-Cl gels(Bio-Rad), electroblotted onto PVDF membranes, and stainedwith Ponceau red to confirm transfer. Membranes were blockedwith 5% nonfat dry milk and 2.5% BSA in TBST for 1 h at roomtemperature followed by consecutive 1-h incubations at roomtemperature with primary antibody in TBST � 1.5% BSA andHRP-conjugated secondary antibody in TBST � 1% nonfat drymilk. Primary antibodies used for western blot analysis weredirected against Noggin and Dkk-1 (Table S2). Protein bandswere visualized by chemiluminescence (ECL Plus Western BlotAnalysis Detection Kit; GE Healthcare).

1. Pankratz MT, et al. (2007) Directed neural differentiation of human embryonic stemcells via an obligated primitive anterior stage. Stem Cells 25:1511–1520.

2. Gamm DM, et al. (2008) Regulation of prenatal human retinal neurosphere growth andcell fate potential by retinal pigment epithelium and Mash1. Stem Cells 26:3182–3193.

3. Gamm DM, et al. (2008) A novel serum-free method for culturing human prenatalretinal pigment epithelial cells. Invest Ophthalmol Vis Sci 49:788–799.

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Fig. S1. Repression of neural and eye field fate specification by BMP4 and Wnt3A. (A) In the absence of exogenous Wnt and BMP antagonists, cells in typicalhESC neuroepithelial colonies at day 10 of differentiation were tightly packed together and individual cells were nearly indistinguishable. (B) When 100 ng/mLBMP4 and Wnt3A were added to cultures at the onset of differentiation, hESCs adopted altered, non-neuroepithelial morphologies by day 10.

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Fig. S2. Eye field specification is dependent upon FGF signaling. RT-PCR showing complete loss of Rx and Pax6 gene expression at day 10 of differentiationin the presence of 10 �M SU5402.

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Fig. S3. Quantification showing the percentage of Chx10� neurospheres (A) and the percentage of Chx10� cells within those spheres (B).

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Fig. S4. Differentiated cells retained an anterior neural phenotype. At 40 days of differentiation, all neurospheres demonstrated expression of the generalneural markers Sox1 (A–C) and �III-tubulin (D–F). (G–I) The anterior neural fate of these cells was determined by the widespread expression of the retina andforebrain transcription factor Otx2. (J) RT-PCR experiments confirmed that these cells expressed both general and anterior neural markers, but did not expressmarkers of other germ layers (alpha-fetoprotein and brachyury), spinal cord (HoxB4), or midbrain (En-1). Insets in (A) and (G) are higher magnification imagesof immunostaining experiments demonstrating nuclear localization of the signal.

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Fig. 5 Schematic of the timing of retinal lineage marker expression during hESC differentiation in comparison to normal human retinal development (22, 23).

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Fig. S6. Quantitative RT-PCR analysis of Pax6(�5a) expression relative to total Pax6 message in differentiating hESC-derived neurosphere cultures. Values areexpressed as fold change relative to cultures at day 4 of differentiation.

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Fig. S7. Schematic of the differentiation protocol used to generate cells of a retinal lineage from human iPS cells.

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Fig. S8. Expression of eye field characteristics in differentiating IMR90–4 iPS cells. After 10 days of differentiation, iPS cells coexpressed Pax6 with eye fieldand anterior neuroepithelial transcription factors such as Lhx2 (A) and Otx2 (B), respectively. (C) Eye field colonies expressed the definitive neural marker Sox1.(D) RT-PCR over the first 16 days of differentiation demonstrated the expression of a full complement of eye field transcription factors, as well as neuroepithelialmarkers.

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Table S1. Primers used for RT-PCR

Gene amplified Forward Reverse Size, bp

�-fetoprotein AGA ACC TGT CAC AAG CTG TG GAC AGC AAG CTG AGG ATG TC 676��Actin (qPCR) GCG AGA AGA TGA CCC AGA TC CCA GTG GTA CGG CCA GAG G 103Bestrophin ATT TAT AGG CTG GCC CTC ACG GAA TGT TCT GCC GGA GTC ATA AAG CCT 359Brachyury ACC CAG TTC ATA GCG GTG AC CAA TTG TCA TGG GAT TGC AG 392Chx10 ATT CAA CGA AGC CCA CTA CCC AGA ATC CTT GGC TGA CTT GAG GAT GGA 229Chx10 (qPCR) GGC GAC ACA GGA CAA TCT TTA TTC CGG CAG CTC CGT TTT C 122Crx TAT TCT GTC AAC GCC TTG GCC CTA TGC ATT TAG CCC TCC GGT TCT TGA 253Dkk-1 (qPCR) AGC ACC TTG GAT GGG TAT TCC AGA ACA CAA TCC TGA GGC ACA GTC TGA 114En-1 CCC TGG TTT CTC TGG GAC TT GCA GTC TGT GGG GTC GTA TT 162GAPDH (23 cycles) ACC ACA GTC CAT GCC AT CAC TCC ACC ACC CTG TTG CTG TA 450HoxB4 (55°C) GCA AAG AGC CCG TCG TCT AC CGT GTC AGG TAG CGG TTG TA 160Lhx2 CAA GAT CTC GGA CCG CTA CT CCG TGG TCA GCA TCT TGT TA 284Mitf TTC ACG AGC GTC CTG TAT GCA GAT TTG CAA AGC AGG ATC CAT CAA GCC 106Nanog CAA AGG CAA ACA ACC CAC TT TCT GCT GGA GGC TGA GGT AT 158Noggin (qPCR) CCA TCA TTT CCG AGT GCA AGT GCT AAG CTA GGT CTC TGT AGC CCA GAA 189Oct4 CGA GCA ATT TGC CAA GCT CCT GAA TTC GGG CAC TGC AGG AAC AAA TTC 324Oct4 (qPCR) GTG GAG GAA GCT GAC AAC AA ATT CTC CAG GTT GCC TCT CA 120Opsin TAC CTG GAC CAT TGG TAT TGG CGT TAA GTC CAG CCC ATG GTT ACG GTT 379Otx2 CAA CAG CAG AAT GGA GGT CA CTG GGT GGA AAG AGA GAA GC TG 429Pax6 CGG AGT GAA TCA GCT CGG TG CCG CTT ATA CTG GGC TAT TTT GC 300(�5a)258(�5a)Pax6 (qPCR) AGT GAA TCA GCT CGG TGG TGT CTT TGC AGA ATT CGG GAA ATG TCG CAC 120Pax6(�5a) (qPCR) CTC GGT GGT GTC TTT GTC AAC ACT TTT GCA TCT GCA TGG GTC 130RPE65 GCC CTC CTG CAC AAG TTT GAC TTT AGT TGG TCT CTG TGC AAG CGT AGT 259Rx GAA TCT CGA AAT CTC AGC CC CTT CAC TAA TTT GCT CAG GAC 279Rx (qPCR) AGC GAA ACT GTC AGA GGA GGA ACA TCA TGC AGC TGG TAC GTG GTG AAA 81Six3 (55°C) CGA GCA GAA GAC GCA TTG CTT CAA CGG CCT TGG CTA TCA TAC ATC ACA 394Six6 ATT TGG GAC GGC GAA CAG AAG ACA ATC CTG GAT GGG CAA CTC AGA TGT 385Sox1 CAA TGC GGG GAG GAG AAG TC CTC TGG ACC AAA CTG TGG CG 464Sox2 (55°C) CCC CCG GCG GCA ATA GCA TCG GCG CCG GGG AGA TAC AT 448Tll ATG GCA AAT TCT GTG GCG CTG AAG GCG CTG ATT TCC CAA GTG CAT TCT 352

All primer sets listed were run for 30 cycles at an annealing temperature of 60°C unless otherwise noted. Quantitative RT-PCR primers were run for 40 cycles.

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Table S2. Primary antibodies used for immunocytochemistry and Western analysis

Antibody Type Source Dilution

�III tubulin Rabbit polyclonal Covance 1:100Chx10 Goat polyclonal Santa Cruz 1:200Crx Mouse monoclonal Abnova 1:100Dkk1 Mouse monoclonal Upstate Biotechnology 1:500Lhx2 Goat polyclonal Santa Cruz 1:200Mitf Mouse monoclonal Neomarkers 1:50Noggin Mouse Monoclonal Chemicon 1:2,000Oct4 Mouse polyclonal Santa Cruz 1:1,000Opsin, red/green Rabbit polyclonal Chemicon 1:5,000Otx2 Goat polyclonal R & D Systems 1:2,000Pax6 Mouse monoclonal Developmental Studies Hybridoma Bank 1:50Recoverin Rabbit polyclonal Chemicon 1:2,000Rx Rabbit polyclonal Abcam 1:1,000Sox1 Goat polyclonal R & D Systems 1:1,000ZO-1 Rabbit polyclonal Zymed 1:100

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