Differentiation of iPS-RPE cells from a patient with clinical anophthalmia

Abstract

Purpose: Mutations in the Stra6 gene have been implicated in a broad spectrum of embryonic developmental abnormalities including cardiac and pulmonary defects, mental retardation and anophthalmia. We have produced induced pluripotent stem (iPS) cells from a patient with isolated clinical anophthalmia as a result of a 3 base-pair in-frame deletion in the coding region of Stra6. Here, we examine their potential to differentiate into retinal pigment epithelial (RPE) cells in vitro.

Methods: Fibroblast cells isolated from the patient were reprogrammed using a Dox inducible lentiviral vector containing Oct4, Klf4, Sox2 and c-myc in a polycistronic cassette. iPS cells were encouraged to differentiate towards RPE cell lineage by removal of bFGF from the culture medium. Differentiated cells were purified and analysed by PCR, immunocytochemistry, electron microscopy and phagocytosis assays. Cells were transplanted into the subretinal space of the RCS rat to examine function in vivo.

Results: iPS cells from patient with clinical anophthalmia differentiate into RPE cells. Purified cells form a pigmented monolayer, with classic cobblestone morphology, which expressed RPE cell markers. Stra6 was expressed by cells but was not localised to the membrane. RPE were highly polarised with a transepithelial resistance higher than control cells. Cells were able to phagocytose photoreceptor outer segments in vitro and in vivo, however few cells remained in the subretinal space 10 days following transplantation.

Conclusions: These data suggest that although Stra6 is required for normal eye development, it is not essential for RPE cell differentiation in vitro. These studies also suggest that iPS technology can be used to create patient specific cells, which may not have developed in the patient themselves.

Keywords: 701 retinal pigment epithelium • 721 stem cells • 500 differentiation