Improving the efficiency of RPE cell differentiation from rhesus macaque iPS- and SCNT-derived stem cell lines

Abstract

Purpose: Stem cell-derived retinal pigment epithelium (RPE) holds promise for treating age-related macular degeneration and other retinal diseases. Somatic cell nuclear transfer (SCNT) and induced pluripotency (iPS) technologies make possible the production of patient-specific RPE cells. A variety of protocols have successfully generated RPE from human iPS cell lines. The purpose of this project is to achieve high efficiency RPE differentiation in rhesus macaque iPS and SCNT cells to provide sources of autologous cells for transplantation and in vitro characterization.

Methods: Rhesus macaque iPS and SCNT cells were grown on mitotically inactivated mouse embryonic fibroblasts. Confluent stem cell cultures were either allowed to spontaneously differentiate, co-cultured with PA6 stromal cells, or differentiated to embyroid bodies in suspension. In addition, combinations of small molecules and growth factors were applied to stem cells in each of these conditions. The resulting pigmented cells were tested for expression of key RPE-associated genes by (RT)PCR and immunocytochemistry.

Results: Spontaneously differentiating SCNT cells did not produce pigmented colonies, whereas iPS cells produced few and small patches of RPE cells. Co-cultures of iPS cells with PA6 feeder cells generated RPE cells at efficiency too low for isolation and expansion. SCNT cells cultured with this method produced cells with epithelial-like morphology but without pigmentation. Suspension culture of SCNT lines resulted in embyroid bodies containing less than 1% pigmented cells. In contrast, a specific small molecule and growth factor regime generated large numbers of iPSC-derived RPE cells that tested positive for RPE-associated markers including RPE65, CRALBP, MITF, PMEL17, Bestrophin, ZO-1 and PEDF. It appeared more difficult to induce RPE differentiation in SCNT lines.

Conclusions: In our experience, several standard protocols for successful differentiation of human RPE cells had low efficiency in rhesus iPS or SCNT lines. A new treatment regime improved efficiency of differentiation from rhesus iPS cells many-fold, yielding cells with the morphology and gene expression signature of RPE. These cells will be delivered subretinally in rhesus monkeys to provide preclinical models of allograft and autograft RPE transplantation and to compare the responses of the retina and immune system to the transplanted cells.