In-depth characterisation of retinal pigment epithelium (RPE) cells derived from human induced pluripotent stem cells (iPSC)

Abstract

Purpose: To establish and comprehensively characterise retinal pigment epithelium (RPE) cells derived from adult human dermal fibroblasts via induced pluripotent stem cell (iPSC) technology.

Methods: Adult human dermal fibroblast cultures were established from skin biopsy material and subsequently reprogrammed following polycistronic lentiviral transduction with OCT3/4, Sox2, Klf4 and l-Myc. Chromosomal integrity was assessed by karyotyping. RPE cell differentiation was achieved by induction with RPE medium enriched for nicotinamide and Activin A. After 8 weeks, pigmented clusters of RPE cells were manually excised and subcultured. Human iPSCs were characterised by RT-PCR expression of specific stem cell markers and immunofluorescence. IPSC-derived RPE cells were characterised by RT-PCR expression of mature RPE markers, confocal microscopy, scanning electron microscopy (SEM) and functional analysis, the latter including feeding experiments with porcine photoreceptor outer segments (POS) and measurements of transepithelial resistance (TER).

Results: Fibroblast-derived human iPSCs showed typical morphology and regular karyograms. Furthermore, they revealed distinctive stem cell marker properties based on RNA- and protein-expression profiling. Subsequently, human iPSCs were differentiated into pigmented clusters reminiscent of RPE cells. These cells maintained typical hexagonal RPE-morphology during subcultivation. Starting at passage 6 replicative senescence increased. RNA expression of mature PRE markers RPE65, RLBP and BEST1 were found in comparison to human iPSCs. Confocal microscopy demonstrated localisation of BEST1 at the basolateral plasma membrane while SEM demonstrated typical microvilli at the apical side of RPE cell. With regard to functional aspects, iPSC-derived RPE cells phagocytosed and shredded POS. Finally, TER measurements showed a significant increase and maintained high levels of TER indicating functional formation of tight junctions.

Conclusions: Our data demonstrate the successful reprogramming of human adult skin biopsy-derived fibroblasts to iPSCs and their differentiation to RPE cells structurally and functionally indistinct from native RPE cells.

Keywords: 701 retinal pigment epithelium • 721 stem cells