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Additional Supporting Information may be found in the online version of this article.

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STEM_674_sm_SuppInfo.pdf134KSupporting Information
STEM_674_sm_SuppFig1.pdf1172KFig. S1. Differential expression of transcription factor genes in vesicle-like structures and nonvesicular spheres. Transcription factor genes with the highest relative fold differences in expression between populations of vesicle-like structures and nonvesicular spheres at day 20 of differentiation were determined by comparative microarray analysis. Transcription factors that were more highly expressed in vesicle-like structures are presented on the right side of the graph as red bars, whereas those that were more highly expressed in nonvesicular spheres are presented on the left side of the graph as blue bars.
STEM_674_sm_SuppFig2.pdf7180KFig. S2. Distribution of neuroretinal cell types within OV-like structures cultured long-term. Sections of OV-like structures cultured for a total of 70 days were immunostained using primary antibodies directed against βIII-TUBULIN (A), BRN3 (B) or RECOVERIN (C-D). βIII-TUBULIN + and BRN3+ cells (retinal ganglion cells) were commonly localized at the periphery of these structures or surrounding neural rosettes (A-B). In contrast, RECOVERIN+ cells (photoreceptor-like cells) were often found internally in clusters (C) or linear formations (D).
STEM_674_sm_SuppFig3.pdf5727KFig. S3. Comparison of RPE derived from hiPSCs, hESCs, and human prenatal eyes. Confocal images demonstrating expression of EZRIN (A) and BESTROPHIN (B) in day 70 differentiated IMR90-4 hiPSC-RPE. The orthogonal views located beneath panels A and B show the characteristic apical and basolateral expression of EZRIN and BESTROPHIN, respectively. (C) qPCR analysis comparing RPE-associated gene expression in WA09 hESC-RPE and IMR90-4 hiPSC-RPE relative to day 94 human fetal RPE. (D) RPE derived from these three sources demonstrated an appropriate increase in [Ca2+]i in response to ATP stimulation (gray bar), as determined by Fura-2 AM epiflourescence imaging. (E) Bar graph depicting the maximal [Ca2+]i responses to ATP stimulation for RPE derived from all three sources (mean ± SEM; n=3).
STEM_674_sm_SuppFig4.pdf1743KFig. S4. Differential expression of anterior neural/eye field transcription factors across human pluripotent stem cell lines. After 10 days of differentiation, the expression of selected anterior neural/eye field transcription factors was determined via qPCR. Data is expressed relative to the lowest expressing line (Lenti iPSC #2).
STEM_674_sm_SuppFig5.pdf3910KFig. S5. Generation of retinal phenotypes from hiPSC-derived OV-like structures. hiPSC-derived OV-like structures yielded progeny expressing markers of specific retinal cell types, including CRX+/RECOVERIN+ photoreceptors (A) and BRN3+ retinal ganglion cells (B). Images shown are from cultures differentiated for 80 days.
STEM_674_sm_SuppFig6.pdf8341KFig. S6. Characterization of gyrate atrophy hiPSCs. hiPSCs derived from a patient with gyrate atrophy (GA) expressed numerous pluripotency-related genes (A-C). Following transplantation into SCID mice (Yu et al., 2007), GA-hiPSCs produced teratomas containing derivatives of all three germ layers (D-F). GA-hiPSCs retained the patient's causative gene mutation (G) and maintained a normal karyotype (H). In panel G, the upper and lower chromatograms present sequencing data from wildtype WA01 hESCs and (A226V)OAT GA-hiPSCs, respectively. The shaded boxes highlight the G>A transition in the antisense strand of (A226V)OAT GA-hiPSCs, which corresponds to the C>T transition at nucleotide 677 in exon 7 of the sense strand [alanine (GCG) to valine (GTG)].
STEM_674_sm_SuppTab1.pdf52KSupplemental Table 1. Primary antibodies used for immunocytochemistry and flow cytometry.
STEM_674_sm_SuppTab2.pdf87KSupplementary Table 2. Primers used for RT-PCR and quantitative RT-PCR.

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