Ueda Kaori, Onishi Akishi, Ito Shin-Ichiro, Nakamura Makoto, Takahashi Masayo
Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Kobe, Hyogo, 650-0047, Japan; Division of Ophthalmology, Department of Surgery, Kobe University Graduate School of Medicine, Kobe, 650-0017, Japan.
Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Kobe, Hyogo, 650-0047, Japan.
Biochem Biophys Res Commun. 2018 Jan 22;495(4):2595-2601. doi: 10.1016/j.bbrc.2017.12.092. Epub 2017 Dec 20.
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.
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.
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.
By modifying culture conditions in the SFEBq protocol, we obtained rod-dominated 3D retinas and S- and M-opsin expressing 3D retinas.
在特定培养基条件下，三维视网膜类器官可从胚胎干细胞/诱导多能干细胞（ES/iPS细胞）分化而来。我们改进了具有快速重聚集功能的类胚体样聚集体的无血清悬浮培养（SFEBq）程序，以获得表达更多视杆光感受器以及S-和M-视锥视蛋白的视网膜类器官。
在光感受器发育过程中，将从小鼠Nrl-eGFP iPS细胞分化而来的视网膜类器官培养于各种培养基中。为促进视杆光感受器发育，将类器官维持在含有9-顺式视黄酸（9cRA）的培养基中。为获得表达M-视蛋白的视网膜类器官，我们在添加了T3、BMP4和DAPT的1%胎牛血清（FBS）培养基中进行培养。进行切片免疫组织化学以观察光感受器标志物的表达。
在暴露于9cRA的三维（3D）视网膜中，视紫红质表达更早，S-视锥视蛋白受到抑制。我们可以在含有1% FBS的培养基中将3D视网膜培养至发育天数（DD）35。3D视网膜表达视紫红质、S-和M-视蛋白，但大多数视锥光感受器仅表达S-或M-视蛋白。
通过修改SFEBq方案中的培养条件，我们获得了以视杆为主的3D视网膜以及表达S-和M-视蛋白的3D视网膜。
