Kaori Ueda1, Akishi Onishi2, Shin-Ichiro Ito3, Makoto Nakamura4, Masayo Takahashi3. 1. 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. 2. Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Kobe, Hyogo, 650-0047, Japan. Electronic address: aonishi@cdb.riken.jp. 3. Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Kobe, Hyogo, 650-0047, Japan. 4. Division of Ophthalmology, Department of Surgery, Kobe University Graduate School of Medicine, Kobe, 650-0017, Japan.
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.
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 mouseNrl-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.
Authors: Arwin Shrestha; Brittany N Allen; Luke A Wiley; Budd A Tucker; Kristan S Worthington Journal: J Ocul Pharmacol Ther Date: 2019-08-16 Impact factor: 2.671
Authors: Evgenii Kegeles; Anton Naumov; Evgeny A Karpulevich; Pavel Volchkov; Petr Baranov Journal: Front Cell Neurosci Date: 2020-07-03 Impact factor: 5.505
Authors: Laura R Bohrer; Luke A Wiley; Erin R Burnight; Jessica A Cooke; Joseph C Giacalone; Kristin R Anfinson; Jeaneen L Andorf; Robert F Mullins; Edwin M Stone; Budd A Tucker Journal: Genes (Basel) Date: 2019-04-05 Impact factor: 4.096
Authors: Nicholas E Stone; Andrew P Voigt; Robert F Mullins; Todd Sulchek; Budd A Tucker Journal: Stem Cells Transl Med Date: 2021-06-22 Impact factor: 6.940