The Developmental Stage of Adult Human Stem Cell-Derived Retinal Pigment Epithelium Cells Influences Transplant Efficacy for Vision Rescue

Abstract

Age-related macular degeneration (AMD) is a common cause of central visual loss in the elderly. Retinal pigment epithelial (RPE) cell loss occurs early in the course of AMD and RPE cell transplantation holds promise to slow disease progression. We report that subretinal transplantation of RPE stem cell (RPESC)-derived RPE cells (RPESC-RPE) preserved vision in a rat model of RPE cell dysfunction. Importantly, the stage of differentiation that RPESC-RPE acquired prior to transplantation influenced the efficacy of vision rescue. Whereas cells at all stages of differentiation tested rescued photoreceptor layer morphology, an intermediate stage of RPESC-RPE differentiation obtained after 4 weeks of culture was more consistent at vision rescue than progeny that were differentiated for 2 weeks or 8 weeks of culture. Our results indicate that the developmental stage of RPESC-RPE significantly influences the efficacy of RPE cell replacement, which affects the therapeutic application of these cells for AMD.

Keywords: AMD; RPE; RPE transplant efficacy; developmental stage; differentiation stage; macular degeneration; retinal pigment epithelium; stem cell; transplant; vision rescue.

Figure 1

RPESC-RPE Mature over 8 Weeks in Culture (A) Passage 0 (P0) RPE cells dissociated from donor cadaver eyes were expanded in culture, re-plated, re-expanded, and then frozen at passage 1 (P1). (B) Banked P1 cells were thawed and cultured to obtain passage 2 (P2) cells at 2-, 4-, or 8-week stages. Representative phase and immunostained images are shown for OTX2, MITF, or ZO-1 expression. Insets show digitally zoomed high magnification images. Scale bar, 50 μm. (C) The fraction of cells expressing Ki67 (Ki67+/DAPI) at 2, 4, and 6/7 weeks. One-way ANOVA (p < 0.01) followed by Tukey's multiple comparison test (2 versus 4, p = 0.0006; 2 versus 6/7, p = 0.0003; 4 versus 6/7, not significant). (D) Secretion of VEGF-A (left) and PEDF (right) from apical and basal surfaces on a transwell membrane also rise during the 8-week RPE maturation period.

Figure 2

Phagocytic Activity of RPESC-RPE with Maturation (A–D) RPESC-RPE cultured on transwell membrane (A) for 2–3 (B), 4–5 (C), or 7–8 weeks (D) were challenged with FITC-labeled POS (B–D). Representative confocal central image stacks show the distribution of internalized POS in green. ZO-1 labeling (red) indicates tight junctions. (E and F) Quantification shows the percentage of cells with internalized POS (E) and the amount of internalized POS per area (F) after 5 hr of POS exposure, expressed as mean ± SEM from three different cell lines for each stage of maturation (shown in weeks, x axes). Asterisks indicate significant differences between groups indicated by horizontal bars; ^∗ = p < 0.05 by ANOVA. Scale bar, 20 μm.

Figure 3

RPESC-RPE Transplantation Photoreceptor Rescue (A) H&E stained retinal sections from wild-type Long Evans (WT), and P100 unoperated (Unop) or RPESC-RPE transplanted (Cells) RCS rat retinas. White arrows indicate loss of ONL. (B and C) Quantification of ONL rescue after RPESC-RPE transplantation into the temporal region of the eye. (B) Matrix and (C) graphical representations of DAPI-stained nuclei counts across the nasal-temporal and inferior-superior axes. Rows contain the average number of nuclei over ∼100–150 μm intervals. Columns contain counts from slides spaced at 200 μm intervals. cb, ciliary body; onh, optic nerve head. (B) Average counts from an RPESC-RPE injected eye. (C) Comparison of the number of rescued photoreceptor nuclei in 17 rats after subretinal injection with 2, 4, or 7 week RPESC-RPE in the temporal region (bracket in B). Three donor lines were used as follows: 228, 4 weeks, 2; 228, 7 weeks, 3; 229, 2 weeks, 3; 229, 4 weeks, 5; 230, 4 weeks, 4. Error bars indicate SD; no significant difference was observed between the 2-, 4-, and 7-week developmental stages tested.

Figure 4

Stage-Dependent RPESC-RPE Transplantation Vision Rescue (A) RPESC-RPE cultured for 2, 4, or 7–8 weeks were transplanted into 55 RCS rats. (B) Spatial frequency thresholds were measured (cycles/degree) at P90 after transplant of 50,000 line 229 RPESC-RPE grown for 2–8 weeks. Cells differentiated for 3, 4, or 8 weeks rescued vision more effectively than 2-week cells or unoperated and BSS injected controls (^∗ = p < 0.05 compared with BSS control). Other significant differences are: unoperated versus 3, 4, and 8 weeks, p < 0.01; and 2 versus 4 weeks, p < 0.05. (C) Line 228 and 230 data at 4 and 7 weeks compiled from five experiments. ^∗ = p < 0.05 indicates comparison with BSS controls. Other significant comparisons are: unoperated versus 228 at 4 and 7 weeks and 230 at 4 weeks, p < 0.01; 228 at 4 and 7 weeks versus 230 at 8 weeks, p < 0.01; and 230 at 4 weeks versus 230 at 7 weeks, p < 0.01. Statistical analysis was a Kruskal-Wallis test (p < 0.0001) followed by Dunn's multiple comparisons test and correction by the two-stage Benjamini, Hochberg, Yekutieli false discovery rate method. (D) To compare 4 week cells with 7/8 week cells, data from (B) and (C) were grouped and analyzed using a Mann-Whitney test, which indicated that 4-week cells rescued vision more effectively than 7- to 8-week cells (p < 0.0001).