Supplementary Materials Supplemental Shape 1 Expression of pluripotent markers by human iPSCs

Supplementary Materials Supplemental Shape 1 Expression of pluripotent markers by human iPSCs. two groups: normal (100??1.43) vs AMD (105.6??7.38), = 0.476. ns = no significance. SCT3-9-364-s002.tif (1.2M) GUID:?AA2F7D19-FB25-420F-927B-6288BED030E4 Supplemental Figure 3 Flow cytometry analysis and percentage of cells containing phagocytized FITC\labeled bovine rod photoreceptor outer segments (ROS). Representative scatter plot and the histograms are used to display data. iPSC\derived RPE negative control (A). FITC\labeled photoreceptor outer segments were detected in RPE lines derived from individuals with no history of AMD (normal, n = 3) (B) or AMD patients (2 atrophic and 2 exudative) (C). No significant difference was observed in iPSC\derived RPE between AMD or controls (D). Normal (10.38%??0.81) vs atrophic AMD (11.10%??1.36), = 0.63; normal (10.38%??0.81) vs exudative AMD (9.17%??0.76), = 0.31; atrophic AMD (11.10%??1.36) vs exudative AMD (9.17%??0.76), = 0.23. SCT3-9-364-s003.tif (476K) GUID:?574CD07C-4477-466A-A39B-3840139BE7DB Supplemental Table 1 Human iPSCs from 8 donors with age\related macular degeneration (AMD) or no history of AMD. SCT3-9-364-s004.docx (14K) GUID:?39AE0C7A-1A8D-4AA0-8E78-033207614939 Supplemental Table 2 List of antibodies used for iPSC and RPE cell markers SCT3-9-364-s005.docx (14K) GUID:?479B9F61-6492-4E38-9714-15175430A359 Supplemental Table 3 RPE Azacyclonol marker gene expression in normal and AMD iPSC\derived RPE cells SCT3-9-364-s006.docx (15K) GUID:?27E6587F-6162-422C-967F-EE6C7511AE6A Supplemental Table 4 Measurement of mitochondrial function in iPSC\derived RPE cells (individual lines). SCT3-9-364-s007.docx (14K) GUID:?65B89699-FEEB-44B2-9420-46D9326524B4 Supplemental Table 5 Complement\related gene expression in normal and AMD iPSC\derived RPE cells cultured on nitrite\modified ECM SCT3-9-364-s008.docx (18K) GUID:?5ED5C4C0-FBC0-4EF9-B2F4-E3ABCB19DB35 Data Availability StatementThe data that support the findings of this study are openly available in in the NCBI Gene Expression Omnibus and are accessible through GEO Series accession number “type”:”entrez-geo”,”attrs”:”text”:”GSE125564″,”term_id”:”125564″GSE125564. Abstract Modeling age\related macular degeneration (AMD) is challenging, because it is a multifactorial disease. To focus on interactions between the retinal pigment epithelium (RPE) and Bruch’s membrane, we generated RPE from AMD patients and used an altered extracellular matrix (ECM) that models aged Bruch’s membrane. Induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from AMD patients or age\matched (normal) controls. RPE derived from Azacyclonol iPSCs were analyzed by morphology, marker expression, transepithelial electrical resistance (TER), and phagocytosis of rod photoreceptor outer segments. Cell viability and connection was examined on nitrite\customized ECM, a typical changes of aged Bruch’s membrane. DNA microarrays with hierarchical clustering and evaluation of mitochondrial function had been utilized to elucidate feasible systems for the noticed phenotypes. Differentiated RPE displayed cell\particular markers and morphology. The TER and Azacyclonol phagocytic capability had been identical among iPSC\produced RPE cultures. Nevertheless, specific clusters were discovered for the transcriptomes of control and AMD iPSC\derived RPE. AMD\produced iPSC\RPE downregulated genes in charge of metabolic\related pathways and cell connection. AMD\derived iPSC\RPE exhibited reduced mitochondrial respiration and ability to attach and survive on nitrite\modified ECM. Cells that did attach induced the expression of Rabbit Polyclonal to NCAPG2 complement genes. Despite reprogramming, iPSC derived from Azacyclonol AMD patients yielded RPE with a transcriptome that is distinct from Azacyclonol that of age\matched controls. When challenged with an AMD\like modification of Bruch’s membrane, AMD\derived iPSC\RPE activated the complement immune system. value .05 was considered statistically significant. Analysis of variance (ANOVA) empirical Bayes (eBayes) method adjusted statistical values, which is suitable for small sample sizes, were used for calculation/analysis with Transcriptome Analysis Console (TAC; Thermo Fisher Scientific) for microarray studies. Expression level changes greater than 1.5\fold and adjusted value .05 are considered statistically significant. 3.?RESULTS 3.1. Differentiation of human iPSCs into RPE cells Reprogrammed iPSCs expressed OCT4, SOX2, stage\specific embryonic antigen 4 (SSEA\4), and keratin sulfate\associated antigens\1\60 (TRA\1\60) (provided as Supplemental Physique S1), indicating the pluripotency of these cells. As described in the Methods section, iPSCs from fibroblasts were induced to form embryoid bodies (EBs) (Physique ?(Physique1A\C).1A\C). Attached EBs then formed neural rosettes before RPE\like cells appeared in the culture (Physique ?(Figure1D).1D). At approximately 45?days, a hexagonal pigmented monolayer of RPE cells formed in culture (Physique ?(Physique1E,F).1E,F). These iPSC\derived RPE cells expressed RPE markers including the visual cycle protein retinal pigment epithelium\specific 65?kDa protein (RPE65), cellular retinaldehyde\binding protein (CRALBP), ezrin, and tight junction protein zonula occludens\1 (ZO\1) (Physique ?(Physique11G). Open in a separate window Physique 1 Differentiation of human\induced pluripotent stem cell (iPSC)\derived retinal pigment epithelial (RPE) cells from donor fibroblasts. Fibroblasts (A) were reprogrammed into an undifferentiated.