Soluble Factors Secreted by Fibroblast Feeder Cells Induce Retinal Pigment Epithelium Differentiation from Human Pluripotent Stem Cells

Abstract

Purpose: : Human pluripotent stem cells (hPSC) are a promising cell source for developmental studies, drug development, and treatment of various degenerative diseases. These cells can be used to derive retinal pigment epithelium (RPE) cells, which are involved in vision-threatening diseases that affect millions of people worldwide. Optimization of an efficient RPE differentiation method would significantly facilitate the development of clinical applications. Identification of growth factors that enhance differentiation is an important yet time-consuming step in this process. There are two types of fibroblast cells that are commonly used as feeder cells in hPSC culture - human foreskin fibroblasts (hFF) and mouse embryonic fibroblasts (mEF). The aim of this study was to compare the inductive effects of soluble factors secreted by hFF and mEF on differentiation of RPE cells from hPSC.

Methods: : For the entire duration of the study, hPSC were differentiated in cell culture media conditioned by fibroblasts (hFF-CM and mEF-CM), as well as the non-conditioned differentiation medium (RPEbasic). The extent of differentiation was evaluated visually by following pigmentation patterns. Moreover, expression of several eye field and RPE precursor genes and proteins during differentiation was analyzed. Finally, the functionality of mature hPSC-derived RPE cells was assessed. In addition, secretion of several growth factors by the fibroblasts was analyzed.

Results: : The results of this study show that mature and functional RPE cells were obtained in all of the tested differentiation conditions. Mature cells were pigmented, had the appropriate morphology, expressed RPE-specific genes and proteins, and were functional. RPE differentiation was more efficient in media conditioned by fibroblasts than in RPEbasic. The growth factor secretion analysis showed significant differences between the types of fibroblasts studied.

Conclusions: : Fibroblast-conditioned media were shown to have a positive influence on RPE differentiation, when compared with RPEbasic, despite the fact that mature RPE cells were obtained in all differentiation conditions. Soluble factors secreted by fibroblasts that might act as inductive agents of RPE differentiation are currently under investigation.

Keywords: retinal pigment epithelium • differentiation • growth factors/growth factor receptors