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

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STEM_1414_sm_SuppFigure1.tif1003KFigure S1. Lysosomal mass increases in OECs that are long-term expanded in culture (A) Quantification of cell spreading area shows a significant increase in cytoplasmic area commensurate with late-stage cell passage. (B) Mitotracker and Lysotracker labelling demonstrates higher mitochondrial and lysosomal mass in senescent OECs. (C) Quantitative Analysis shows a significant increase in lysosomal mass when earlypassage OECs were compared to senescent counterparts. P<0.001.
STEM_1414_sm_SuppFigure2.tif1335KFigure S2. Distinct interactomes for early-passage and late-passage OECs (A) VisANT software was used to create transcriptome-based interactome for earlypassage OECs and identified functional clusters for cell cycle, DNA repair, and DNA replication. (B) Late-passage OECs interactome identified cell adhesion, stress response, lysosome, and proteolysis as relevant clusters.
STEM_1414_sm_SuppFigure3.tif2622KFigure S3. OEC senescence is coupled to endothelial differentiation (A) Transcriptome-based heatmap for progenitor and endothelial differentiation markers in three biological replicates for early and late OEC passages. (B) Percentage of cells expressing progenitor markers CD34 and c-kit decreased significantly from early to late passages as shown by the flow cytometric analysis.
STEM_1414_sm_SuppFigure4.tif1194KFigure S4. Inflammatory cytokines as regulators of OEC senescence (A) Ingenuity Pathway Analysis software was used to establish a gene network for transcripts that are significantly upregulated in senescent OECs. This approach identified involvement of many inflammatory cytokines (shown in yellow), including IL1A and IL8. (B) REVIGO software analysis of significantly upregulated transcripts in senescent OECs highlighted cytokine production as a master regulator of OEC senescence because it was located at the centre of the semantic space.
STEM_1414_sm_SuppFigure5.tif205KFigure S5. Inflammatory cytokines are upregulated in senescent OECs Real Time qRT-PCR was performed to compare early-passage and senescent OECs. Results show upregulation of the mRNAs encoding the inflammatory proteins IL1A, IL1B, IL8, and ICAM. Red line marks the baseline for housekeeping gene RPL11. Figure S6. Distinct secretomes in early-passage and senescent OECs Protein profiles were evaluated from the conditioned media of early-passage and senescent OECs by using the human cytokine proteome profiler. Proteins upregulated with senescence are outlined in a red box, proteins downregulated with senescence are outlined in yellow, and proteins that did not change are outlined in blue. Positive controls appeared in top corners and bottom left corner. Negative control appeared in bottom right corner.
STEM_1414_sm_SuppFigure6.tif256KFigure S6. Distinct secretomes in early-passage and senescent OECs Protein profiles were evaluated from the conditioned media of early-passage and senescent OECs by using the human cytokine proteome profiler. Proteins upregulated with senescence are outlined in a red box, proteins downregulated with senescence are outlined in yellow, and proteins that did not change are outlined in blue. Positive controls appeared in top corners and bottom left corner. Negative control appeared in bottom right corner.
STEM_1414_sm_SuppFigure7.tif43KFigure S7. OEC cultures are free of endotoxin Conditioned media from early passage and senescent OECs was assessed for endotoxin levels. According to the FDA, sterile water has levels between 0.25-0.5 EU/ml. Our culture conditions are both below 0.3 EU/ml.
STEM_1414_sm_SuppFigure8.tif250KFigure S8. IL8 knockdown in OECs (A) Representative phase-contrast image of OECs transduced with IL8-shRNA using a lentiviral vector after selection with puromycin. (B) Fluorescence image showing transduced-OECs in green, by using GFP as a vector reporter gene. Quantification revealed a transduction efficiency of 93%. (C) Real time qRT-PCR demonstrated efficient IL8 knockdown when OECs were transduced with IL8-shRNA and compared to control-shRNA. Red dotted line marks baseline for housekeeping gene RPL11.
STEM_1414_sm_SuppFigure9.tif99KFigure S9. IL8 modulates expression of other inflammatory cytokines Quantitative analysis of protein profiles shows that IL8 depletion affects expression of IL6 and MCP-1.
STEM_1414_sm_SuppFigure10.tif86KFigure S10. IL8 depletion decreased DNA damage in ex-vivo expanded OECs Quantitative analysis for 53BP1 foci/nuclei demonstrates that IL8-depleted OECs have less DNA damage compared to control cells. Medina et al. 4 IL8 mediates OEC senescence.
STEM_1414_sm_SuppFigure11.pdf245KFigure S11. shRNA-IL8 OECs triggered a local inflammatory reaction (A) Flat mounted retinas stained with Isolectin (green) labels the retinal vasculature and myeloid cells. OECs delivered into the ischemic retinas were pre-labelled with Qdots (red). (B) Higher magnification showing a vascular tube formed by shRNA-IL8 OECs (red) in the ischemic retina that is infiltrated by numerous myeloid cells (round green cells). Magnification ×20 in A and B.
STEM_1414_sm_SuppFigure12.pdf450KFigure S12. IL8 effects on OEC function (A) Representative images for OEC clonogenic assays to study IL8 effects at different passages (P: Cellular Passage). (B) Quantification and statistical analysis of the percentage of colony formation for OECs exposed to recombinant human IL8. (EP: Early-passage; MP: Midpassage; LP: Late-passage; SEN: Senescent). (C) Representative images of scratch wound assay using OECs from two different passages exposed to 100ng/ml IL8 with magnification ×10. (D) Quantification and statistical analysis of the OEC-migrated area after 8 hours (AU: arbitrary units). (E) Representative images of Matrigel-based 3D tube formation assay using OECs from different passages and 1ng/ml IL8 with magnification ×10. (F) Quantification and statistical analysis of OEC tube length after 72 hours of exposure to different concentrations of recombinant human IL8. (G) Representative images of SA-β Galactosidase assay to identify senescent OECs when exposed to 10 ng/ml IL8 with magnification ×20. (H) Quantification and statistical analysis of the percentage of SA-βGal positive cells after exposure to different concentrations of recombinant human IL8. The error bars indicate standard error of the mean. *p<0.05, **p<0.01, ***p<0.001, ns:not significant.
STEM_1414_sm_SuppInfo.pdf111KSupporting Information
STEM_1414_sm_SuppTable1.pdf66KSupporting Information

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