In-Depth Characterisation of Retinal Pigment Epithelium (RPE) Cells Derived from Human Induced Pluripotent Stem Cells (hiPSC)

Abstract

Induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) has widely been appreciated as a promising tool to model human ocular disease emanating from primary RPE pathology. Here, we describe the successful reprogramming of adult human dermal fibroblasts to iPSCs and their differentiation to pure expandable RPE cells with structural and functional features characteristic for native RPE. Fibroblast cultures were established from skin biopsy material and subsequently reprogrammed following polycistronic lentiviral transduction with OCT4, SOX2, KLF4 and L-Myc. Fibroblast-derived iPSCs showed typical morphology, chromosomal integrity and a distinctive stem cell marker profile. Subsequent differentiation resulted in expandable pigmented hexagonal RPE cells. The cells revealed stable RNA expression of mature RPE markers RPE65, RLBP and BEST1. Immunolabelling verified localisation of BEST1 at the basolateral plasma membrane, and scanning electron microscopy showed typical microvilli at the apical side of iPSC-derived RPE cells. Transepithelial resistance was maintained at high levels during cell culture indicating functional formation of tight junctions. Secretion capacity was demonstrated for VEGF-A. Feeding of porcine photoreceptor outer segments revealed the proper ability of these cells for phagocytosis. IPSC-derived RPE cells largely maintained these properties after cryopreservation. Together, our study underlines that adult dermal fibroblasts can serve as a valuable resource for iPSC-derived RPE with characteristics highly reminiscent of true RPE cells. This will allow its broad application to establish cellular models for RPE-related human diseases.Electronic supplementary materialThe online version of this article (doi:10.1007/s12017-014-8308-8) contains supplementary material, which is available to authorized users.

High-throughput physical phenotyping of cell differentiation

“…There have been studies demonstrating changes in expression of several genes and transcription factors (e.g., cathepsin D, Pax 6, calbindin, PKC-a, and Mitf) during pluripotent stem cell differentiation 34,35 ; many of these contribute to the organization of the cytoskeleton through microfilaments, intermediate filaments, and/or microtubules [36][37][38][39][40][41][42][43] . For example, upregulation of Pax6 or PKC-a during differentiation may stabilize the cytoskeleton 44,45 .…”

Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells

“…To analyze functional aspects of BEST1 in human RPE, we resorted to RPE differentiated from humaninduced pluripotent stem cells. Such cell lines reveal properties largely overlapping those of native cells (38) and were generated from a healthy donor and two macular dystrophy patients harboring the heterozygous mutations BEST1-A243V and BEST1-Q238R, respectively (SI Appendix, Fig. S5 A-F).…”

Langmuir-Schaefer film deposition onto honeycomb porous films for retinal tissue engineering

“…Mature RPE cells are pigmented cells typically characterised by a hexagonal cobblestone-like morphology that form a tightly packed epithelium [8,48]. Right from the observation of these simple features, it is already apparent that the layered and highly organised nanostructure of the LS films (both for the honeycomb film and PET control) favours the preservation of the RPE phenotype, as well as the adhesion and spreading of cells, as compared to the substrates where collagen is randomly adsorbed to the samples.…”