In-depth characterisation of Retinal Pigment Epithelium (RPE) cells derived from human induced pluripotent stem cells (hiPSC)

Neuromolecular Med. 2014 Sep;16(3):551-64. doi: 10.1007/s12017-014-8308-8. Epub 2014 May 7.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Animals
  • Antigens, Differentiation / analysis
  • Cell Differentiation
  • Cell Separation
  • Cells, Cultured
  • Coculture Techniques
  • Cryopreservation
  • Culture Media / pharmacology
  • Epithelial Cells / cytology*
  • Epithelial Cells / metabolism
  • Eye Proteins / biosynthesis
  • Eye Proteins / genetics
  • Female
  • Fibroblasts / cytology
  • Fibroblasts / drug effects
  • Hexadimethrine Bromide / pharmacology
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Induced Pluripotent Stem Cells / drug effects
  • Karyotyping
  • Kruppel-Like Factor 4
  • Lentivirus / physiology
  • Mice
  • Microvilli / ultrastructure
  • Phagocytosis
  • RNA, Messenger / biosynthesis
  • RNA, Messenger / genetics
  • Retinal Pigment Epithelium / cytology*
  • Rod Cell Outer Segment
  • Skin / cytology
  • Sus scrofa
  • Tissue Preservation
  • Vascular Endothelial Growth Factor A / metabolism

Substances

  • Antigens, Differentiation
  • Culture Media
  • Eye Proteins
  • KLF4 protein, human
  • Klf4 protein, mouse
  • Kruppel-Like Factor 4
  • RNA, Messenger
  • VEGFA protein, human
  • Vascular Endothelial Growth Factor A
  • Hexadimethrine Bromide