Generation of three-dimensional retinal organoids expressing rhodopsin and S- and M-cone opsins from mouse stem cells

摘要

Abstract Purpose Three-dimensional retinal organoids can be differentiated from embryonic stem cells/induced pluripotent stem cells (ES/iPS cells) under defined medium conditions. We modified the serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) culture procedure to obtain retinal organoids expressing more rod photoreceptors and S- and M-cone opsins. Methods Retinal organoids differentiated from mouse Nrl-eGFP iPS cells were cultured in various mediums during photoreceptor development. To promote rod photoreceptor development, organoids were maintained in media containing 9- cis retinoic acids (9cRA). To obtain retinal organoids with M-opsin expression, we cultured in medium with 1% fetal bovine serum (FBS) supplemented with T3, BMP4, and DAPT. Section immunohistochemistry was performed to visualize the expression of photoreceptor markers. Results In three-dimensional (3D) retinas exposed to 9cRA, rhodopsin was expressed earlier and S-cone opsins were suppressed. We could maintain 3D retinas up to DD 35 in culture media with 1% FBS. The 3D retinas expressed rhodopsin, S- and M-opsins, but most cone photoreceptors expressed either S- or M-opsins. Conclusion By modifying culture conditions in the SFEBq protocol, we obtained rod-dominated 3D retinas and S- and M-opsin expressing 3D retinas. Highlights ? Modified SFEBq culture enabled the visualization of each photoreceptor subtype. ? 9-cis retinoic acid (9cRA) promoted rod photoreceptor development in the 3D retina. ? 3D retinas could be maintained for a longer period by 1% FBS supplementation.