Toward the defined and xeno-free differentiation of functional human pluripotent stem cell-derived retinal pigment epithelial cells

Abstract

Purpose: The production of functional retinal pigment epithelium (RPE) cells from human embryonic (hESCs) and human induced pluripotent stem cells (hiPSCs) in defined and xeno-free conditions is highly desirable, especially for their use in cell therapy for retinal diseases. In addition, differentiated RPE cells provide an individualized disease model and drug discovery tool. In this study, we report the differentiation of functional RPE-like cells from several hESC lines and one hiPSC line in culture conditions, enabling easy translation to clinical quality cell production under Good Manufacturing Practice regulations.

Methods: Pluripotent stem cells were cultured on human fibroblast feeder cells in serum-free medium. The differentiation toward RPE was induced by removing basic fibroblast growth factor and feeder cells from the serum-free conditions. RPE differentiation was also achieved using xeno-free and defined culture conditions. The RPE cell morphology and pigmentation of the cells were analyzed and the expression of genes and proteins characteristic for RPE cells was evaluated. In vitro functionality of the cells was analyzed using ELISA measurements for pigment epithelium derived factor (PEDF) secretion and phagocytosis of photoreceptor outer segments (POS). The integrity of the generated RPE layers was analyzed using transepithelial electric resistance measurements.

Results: We generated putative RPE cells with typical pigmented cobblestone-like morphology. The expression of RPE-specific markers was confirmed at the gene and protein level. The differentiated cells were able to phagocytose POS and secrete PEDF characteristic of native RPE cells. In addition, cultured cells formed a polarized epithelium with high integrity and exhibited excellent transepithelial electric resistance values, indicating well established, tight junctions. Moreover, we introduced an improved method to generate functional putative RPE cells without xeno-components under defined conditions.

Conclusions: We have developed a progressive differentiation protocol for the production of functional RPE-like cells from hESCs and hiPSCs. Our results demonstrate that putative hESC-RPE and hiPSC-RPE express genes and proteins characteristic for RPE cells, as well as being able to phagocytose POS and secrete PEDF. Furthermore, our results show that RPE-like cells can be differentiated in xeno-free and defined culture conditions, which is mandatory for Good Manufacturing Practice-production of these cells for clinical use.

Figure 1

Differentiation of human pluripotent stem cells toward retinal pigment epithelium cells. A: A schematic representation of retinal pigment epithelium (RPE) cell differentiation during retinal development. B: Reverse transcription (RT)–PCR analysis of typical genes for retinal development expressed during putative RPE differentiation of the human embryonic stem cell (hESC) line Regea 08/023 and human induced pluripotent stem cell (hiPSC) line FiPS 5–7 at sequential time points on D7–D72.

Figure 2

A schematic illustration of implementation of the study. Analyses which were done from the RPEregES condition besides the RPEbasic condition are marked with asterisks (*). Analyses were done from floating aggregate cultures unless marked with gray boxes for adherent culture.

Figure 3

Immunofluorescence staining of human embryonic stem cell (hESC; Regea 08/023)- and human induced pluripotent stem cell (hiPSC; FiPS 5–7)-derived retinal pigment epithelium cells revealing maturation stage after 83 days of differentiation. Cellular retinaldehyde-binding protein (CRALBP) and microphthalmia-associated transcription factor (MITF) localization in A-C: manually selected hESC-RPE cells and D-F: hiPSC-retinal pigment epithelium (RPE) cells. G, I: RPE65 expression in hESC-RPE and J, L: hiPSC-RPE. H, K: For cell morphology, F-actins were stained using phalloidin. Tight junction protein anti-zonula occludens (ZO)-1 and proliferation marker Ki67 localization in M: hESC-RPE cells and N: hiPSC-RPE cells. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Images A-F were taken with an Olympus BX60 microscope (Olympus, Tokyo, Japan) using a 60× oil immersion objective, scale bar 20 μm. Images G-N were taken with an LSM 700 confocal microscope (Carl Zeiss) using a 63× oil immersion objective, scale bar 20 μm.

Figure 4

Retina and anterior neural fold homeobox (RAX) expression by FiPS 5–7 during differentiation. Undifferentiated cells (D0) have been used as a reference sample.

Figure 5

Morphology and gene expression analysis of manually selected and long-term cultured human embryonic stem cell (hESC)-retinal pigment epithelium (RPE; Regea 08/023) and hiPSC-RPE (FiPS 5–7) cells. A: Bright-field micrograph of hESC-retinal pigment epithelium (RPE) and human induced pluripotent stem cell (hiPSC)-RPE cells cultured for 136 days on Collagen IV. The cells have acquired a cobblestone morphology and a high degree of pigmentation, which is typical of RPE cells. Low magnification images were captured with a Nikon Eclipse TE2000-S phase contrast microscope (Nikon Instruments Europe B.V. Amstelveen, The Netherlands) and higher magnification images with an Olympus BX60 microscope (Olympus, Tokyo, Japan) using a 60× oil immersion objective. Scale bar 20 μm. B: Reverse transcription (RT)–PCR analysis showing the expression of optic vesicle, optic cup, RPE, neural crest melanocyte, pluripotent stem cell, mesoderm and endoderm marker genes by undifferentiated cells (D0), human foreskin fibroblast (hFF) feeder cells, and putative RPE cells (D196) from Regea 08/023 and FiPS 5–7 cells. N/A=not analyzed. C: Relative OCT3/4 expression between undifferentiated FiPS 5–7 and putative hiPSC-RPE after 196 days of differentiation.

Figure 6

Phagocytosis of photoreceptor outer segments (POS) and cell membrane polarization of human embryonic stem cell (hESC; Regea 08/023) and human induced pluripotent stem cell (hiPSC; FiPS 5–7)-derived retinal pigment epithelium (RPE) cells. A: Putative hESC-RPE and B: hiPSC-RPE internalize POS (green, arrowheads) for cell morphology; F-actins were stained using phalloidin (red). Vertical confocal sections showing apical localization of Na⁺/K⁺ATPase (green) and basolateral localization of Bestrophin (red) in C: hESC-RPE and D: hiPSC-RPE. Images were taken with an LSM 700 confocal microscope (Carl Zeiss) using a 63× oil immersion objective, scale bar 20 μm.

Figure 7

Differentiation of human pluripotent stem cells toward retinal pigment epithelium (RPE) cells under defined culture conditions, RPEregES. All represented images are from human embryonic stem cell (hESC)-RPE Regea 08/023. A: Reverse transcription (RT)–PCR analysis of typical genes for retinal/ RPE development expressed by undifferentiated hESC (Regea 08/023), human foreskin fibroblast (hFF) feeder cells, and putative hESC-RPE on D7 and D44. Expression of B: Microphthalmia-associated transcription factor (MITF), B: Cellular retinaldehyde-binding protein (CRALBP), and E,G: RPE65 on D83. F: For cell morphology, F-actins were stained using phalloidin. H: Proliferative activity was studied by Ki67 staining together with tight junction protein anti-zonula occludens (ZO)-1 in hESC-RPE. I: Vertical confocal sections showing apical localization of Na⁺/K⁺ATPase (green) and basolateral localization of Bestrophin (red). Nuclei stained with 4',6-diamidino-2-phenylindole (DAPI). Images B-D were taken with an Olympus BX60 microscope (Olympus, Tokyo, Japan) using a 60× oil immersion objective, scale bar 20 μm. Images E-I were taken with an LSM 700 confocal microscope (Carl Zeiss) using a 63× oil immersion objective, scale bar 20 μm.