Stepwise Induction of Cone- and Rod-Photoreceptor Differentiation From Mouse ES Cell-Derived Retinal Progenitors

Abstract

Purpose: : An increasing number of patients suffer from loss of vision due to retinal degenerative diseases. Since photoreceptor impairment is the direct cause of visual loss, the production of photoreceptor would contribute greatly to the research and therapies of these diseases. We previously reported the efficient differentiation of mouse ES cells into retinal progenitors in floating aggregate culture, SFEB/DLFA method. However, the in vitro differentiation of these progenitors into photoreceptors occurs only infrequently unless they are co-cultured with embryonic retinal tissues. The purpose of this study is to elucidate the more effecitive condition of photoreceptor differentiation from mouse ES cells.

Methods: : Mouse ES cells were cultured in floating aggregates (SFEB/DLFA treatment) and the induced neural retinal progenitors were used for further examination.

Results: : Neural retinal progenitors derived from mouse ES cells by SFEB/DLFA treatment were purified by FACS. Crx⁺ photoreceptor precursors were efficiently induced in an aggregate culture of these cells by treatment with a Notch signal inhibitor (DAPT). These cells subsequently produce cone photoreceptors at high frequencies, but the differentiation of rhodopsin⁺ rod cells is less efficient. However, further treatment with Fgfs, shh, taurine, and retinoic acid significantly increases the frequency of rhodopsin⁺ cells.

Conclusions: : We have succeeded in efficient generation of cone and rod photoreceptors from mouse ES cell-derived retinal progenitors by step-wise treatments with soluble factors. The ability to generate photoreceptors efficiently in vitro could be a key to understanding and developing treatments for retinal diseases.

Keywords: photoreceptors • differentiation • regeneration