From: Inhibition of T-Cell Activation by Retinal Pigment Epithelial Cells Derived From Induced Pluripotent Stem Cells Invest. Ophthalmol. Vis. Sci.. 2015;56(2):1051-1062. doi:10.1167/iovs.14-15619 Figure Legend: Establishment of human iPS cells from human skin fibroblasts and terminally differentiated human RPE cells from iPS cells. (A) Summary of establishment of human iPS cells. HD, healthy donor; RP, retinitis pigmentosa; CiRA, Center for iPS Cell Research and Application (Kyoto University). (B) Polymerase chain reaction analysis of integration of episomal vectors in the generated iPS cells (TLHD1). As a positive control, two kinds of DNA were used positive control (PC) lines, including plasmid integrated lines, and the pCXLE-hSK vector (2 ng). (C) Immunofluorescence analysis of expression of the pluripotency markers Oct3 and -4, TRA-1-60, and SSEA-4 in human iPS cells (836B1). Induced pluripotent stem cells were also stained with isotype control (mouse IgG). Cell nuclei were counterstained with DAPI. Scale bar: 200 μm. (D) Retinal pigment epithelium cells induced from iPS cells (836B1) clearly showed polygonal morphology, mostly hexagonal, and contained melanin. (E) Measurement of phagocytosis by iPS-RPE cells. Induced pluripotent stem-RPE cells (836B1) were cocultured with FITC-ROS at 37°C (blue) and analyzed using flow cytometry. Control cells, which were iPS-RPE cells cultured with FITC-ROS at 4°C, were used to obtain baseline fluorescence (green), and iPS-RPE cells without ROS at 37°C were also analyzed (red). (F) Retinal pigment epithelium cell–specific markers MiTF and ZO-1 in iPS-RPE cells (836B1) were detected by immunostaining. Scale bars: 50 μm. (G) Detection of RPE marker genes in iPS-RPE cells. Total RNA was extracted from iPS-RPE cells (836B1, 454E2, and 101G26). Human iPS cells, 836B1, were also prepared as a control. For PCR amplification, cDNA was amplified by using primers for human bestrophin (Best), RPE65, Pax6, TGFβ1, TGFβ2, TGFβ3, Lin28, Nanog, and β-actin. Results indicate the relative expression of these molecules (ΔΔCt: control iPS cells = 1). ND, not detected. Date of download: 10/6/2017 The Association for Research in Vision and Ophthalmology Copyright © 2017. All rights reserved.