Fig 1.
Differentiation and purification of pigmented cells from Nrl-GFP mouse iPSC.
(a) Schematic of the procedure. SFEBq; serum-free floating culture of embryoid body-like aggregates with quick reaggregation, KSR; knockout serum replacement. (b) Appearance of aggregate at differentiation day (DD) 7. (c) Differentiation into retinal structure was confirmed by evaluation of Nrl-GFP at DD26. Optical and fluorescence images are shown. (d) Broken up aggregates. (e) Large cell clumps and sheets were collected. (f and g) Phase-contrast (f) and bright field (g) images of pigmented (P) and non-pigmented (NP) colonies at DD11 are shown. (h) Time-dependent change of relative expression of Rx evaluated by qRT-PCR. Data from three independent experiments are shown in different colors. Each mark indicates the mean value of triplicate evaluations. Tukey-Kramer test. n = 3. (i-l) Phase-contrast images at DD16 (i and j) and at DD29 (k and l) are shown. Magnified images (j and l) show the cobblestone appearance at DD29. (m) Pigmented cell number obtained from 60 aggregates with or without Y27632. Mann-Whitney U test. Data from seven independent experiments are shown. Bar indicates the median values. Scale bars, 500 μm (c-g), 200 μm (b) and 100 μm (i-l). **: p<0.01, *: p<0.05.
Fig 2.
Morphological evaluation of DD29 cells.
(a) Confocal imaging of ZO-1 and P-cadherin in the DD29 cells and PN day10-mouse RPE. The polarized expression pattern of ZO-1 on the apical side of the cells is shown (black arrows). Nuclei were counter-stained with DAPI. (b) Electron microscopy image of the DD29 cells. Magnified views of the areas of the yellow dotted squares show the presence of microvilli (c), intercellular attachment (d), and extracellular matrix (e). (f) Immunocytochemistry of ZO-1 of DD29 cells maintained by MEM / N1 / FBS medium and serum-free RPE medium (SFRM). Most cells maintained by MEM / N1 / FBS medium were in a cuboidal shape and formed a monolayer structure. ZO-1-positive cell clumps (arrows) in a different plane of Z-scan imaging with increased cell sizes was observed when cells were maintained with SFRM. Scale bars: 30um (f), 20 μm (a), 10 μm (b), 2 μm (c) and 0.5 μm (d and e).
Fig 3.
(a and b) Representative photograph of DD29 iPS-RPE. Bright-field photos (a) were analyzed by Image J software to obtain the number of pixels representing the area of less pigmented cells, as shown in the black region surrounded by a yellow line in (b). (c) Proportion of pigmented cells to total area of the images was expressed as a percent. Three wells were evaluated in each experiment, and data from four independent experiments are shown. Bars indicate the mean value. (d) Representative data of flow cytometry analysis evaluated by side-scattering (SSC) parameter and forward-scattering (FSC) parameter. There was a main population (P1) in addition to a subpopulation with relatively low SSC (P2). (e) Bright-field photos of 5000 cells/well from the P1 and P2 populations. (f) Pigmentation of the P1 population was more intense than that of the P2 population. Mann-Whitney U test. n = 12 for ‘P1 population’ group, n = 6 for ‘P2 population’ group. Scale bars: 500 μm (e), 200 μm (a, b). **: p<0.01. Error bars in (f) represent S.D.
Fig 4.
Expression of developmental and functional markers of RPE.
Transcriptional analysis of iPSC (DD0), iPS-RPE (DD29) and control RPE freshly obtained from PN day10-mice is shown. Values were normalized by Gapdh. The expression levels of each marker are presented as triplicate data from three independent experiments with different colors.
Fig 5.
Functional characteristics of mouse iPS-RPE.
(a and b) Two types of pRPE were compared with mouse iPS-RPE. Phase-contrast images of 2 week-cultured pRPE obtained from adult mice (pRPE (2w), a) and 2 day-cultured pRPE obtained from PN day10-mice (pRPE (2d), b) are shown. (c) The expression of RPE functional marker mRNAs in fresh PN day10-RPE, DD29 iPS-RPE, pRPE (2d), and pRPE (2w) is shown. The data for fresh RPE and iPS-RPE are the same as those shown in Fig 4. (d and e) Rod outer segment phagocytosis assay. Phase-contrast image and fluorescent image of iPS-RPE that was co-cultured with or without FITC-ROS and washed by medium are shown (d). Both iPS-RPE and pRPE (2w) phagocytosed FITC-ROS, and the percentage of FITC-positive cells in iPS-RPE was significantly higher than that in pRPE (2w). Tukey-Kramer test. n = 3. (f) Immune surface antigen expression evaluated by flow cytometry. The red line histograms represent isotype control. Numbers in the histogram indicate the percentage of positive cells. (g and h) Percentage of Ki-67 and CD4 double-positive T cells (g) and that of Ki-67 and CD8 double-positive T cells (h) after stimulation by anti-CD3 and anti-CD28 antibody. iPS-RPE significantly suppressed the proliferation of CD4-positive T cells and CD8-positive T cells. Tukey-Kramer test. n = 3. Scale bars: 200 μm (a, b) and 100 μm (d). **: p<0.01, *: p<0.05.