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Figure 1. Human iPSC-Derived Retinal Pigment Epithelial Cells Show Characteristic RPE Morphology

Cryopreserved Human iPSC-Derived Retinal Pigment Epithelial (RPE) Cells were thawed and plated onto Corning® Matrigel®-coated plates at 150,000 cells/cm². RPE cells were maintained in STEMdiff™-XF RPE Maturation Medium supplemented with STEMdiff™-ACF RPE Plating Supplement for 7 days, followed by STEMdiff™-XF RPE Maturation Medium alone thereafter. The cells were incubated at 37°C and subsequently analyzed by brightfield microscopy at various time points. Representative microscopy images show that RPE cells display the expected morphology after more than 63 days of culture.

Figure 2. Human iPSC-Derived Retinal Pigment Epithelial (RPE) Cells Are Mature and Functional at Day 35.

Human iPSC-Derived RPE Cells were thawed and cultured in STEMdiff™-XF RPE Maturation Medium for at least 35 days to demonstrate RPE maturity.

(A) The percentage of RPE cells, derived from two different iPSC lines, expressing PMEL17, CRALBP, EZRIN, and RPE65 was assessed by flow cytometry analysis. Data are reported as mean ± SEM (n = 4).

(B,C) Mature RPE cells exhibit high expression of CRALBP and display extensive tight junctions, as indicated by the localization of ZO-1 and BEST1 at cell boundaries. (D,E) Mature RPE cells are polarized, expressing EZRIN (3D projection) and proteins essential for the visual cycle, such as RPE65. These markers are visualized by fluorescence microscopy.

Figure 3. Human iPSC-Derived Retinal Pigment Epithelial Cells Display Key RPE Functionality

Human iPSC-Derived Retinal Pigment Epithelial (RPE) Cells were cultured on cell culture inserts in STEMdiff™-XF RPE Maturation Medium for 35 days. Apical and basal conditioned media were collected from mature RPE cells, and a sandwich ELISA was performed to quantify vascular endothelial growth factor (VEGF) and pigment epithelium-derived growth factor (PEDF) secretion.

(A) A cross-sectional schematic of the cell insert culture system illustrates the setup. Mature RPE cells, derived from two iPSC lines, secreted higher levels of (B) VEGF basally and (C) PEDF apically, demonstrating correct apicobasal polarity.

(D) Mature RPE cells also generated a strong barrier with high transepithelial resistance (TER). (E) Additionally, mature RPE cells were fed FITC-labeled bovine photoreceptor outer segments (POS) for 4 - 5 hours and efficiently internalized the bovine POS. Data are presented as mean ± SEM.