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Biochem Biophys Res Commun. 2018 Jan 22;495(4):2595-2601. doi: 10.1016/j.bbrc.2017.12.092. Epub 2017 Dec 20.

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

Author information

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.

KEYWORDS:

3D retinal aggregates; Cone photoreceptor; Retinoic acid; Rod photoreceptor; Stem cells

PMID:
29274337
DOI:
10.1016/j.bbrc.2017.12.092
[Indexed for MEDLINE]

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