Differentiation Protocol for 3D Retinal Organoids, Immunostaining and Signal Quantitation

Abstract

Structures resembling whole organs, called organoids, are generated using pluripotent stem cells and 3D culturing methods. This relies on the ability of cells to self-reorganize after dissociation. In combination with certain supplemented factors, differentiation can be directed toward the formation of several organ-like structures. Here, a protocol for the generation of retinal organoids containing all seven retinal cell types is described. This protocol does not depend on Matrigel, and by keeping the organoids single and independent at all times, fusion is prevented and monitoring of differentiation is improved. Comprehensive phenotypic characterization of the in vitro-generated retinal organoids is achieved by the protocol for immunostaining outlined here. By comparing different stages of retinal organoids, the decrease and increase of certain cell populations can be determined. In order to be able to detect even small differences, it is necessary to quantify the immunofluorescent signals, for which we have provided a detailed protocol describing signal quantitation using the image-processing program Fiji.

The Role of Small Molecules and Their Effect on the Molecular Mechanisms of Early Retinal Organoid Development

“…We noticed in the literature that a subset of factors were commonly added together to target a single pathway simultaneously, or influence multiple pathways. For example, dual SMAD inhibition by SB431542 and LDN193189 is used to regulate both the branches of the TGF-β/BMP signaling pathway, and we found that they were added exclusively together in retinal organoid protocols [107,108] (Table 1). The suppression of TGF-β and BMP signaling has also been achieved by SB431542 and dorsomorphin treatment and is used in vitro and in vivo to direct cells into the neural and retinal fates [105,268,269].…”

“…SB431542 is typically added to cultures within the first seven days of cultures (Table 1). It improves the development of specific retinal cell types, such as photoreceptors, both in 2D cultures and 3D retinal organoid models [107][108][109]. COCO is part of the Dan family of TGF-β antagonists [110], and is commonly used as an inhibitor of the BMP branch of the signaling pathway (Figure 3).…”

“…In retinal differentiation, both these small molecules have been used in organoid models to increase the efficiency of retinal generation [127,128]. However, they are usually used in combination with SB431542 as part of a dual SMAD inhibition to inhibit both the TGF-β and BMP sides of the pathway [107,108].…”

“…(C) Alongside stand-alone protocols used solely by a single group (in blue), there were also a small number of manuscripts that did not relate to other methods. All papers included either produced retinal organoids, or were referenced as a method of producing retinal organoids[48,49,74,101,[107][108][109]113,127,[129][130][131]135,[143][144][145][146][148][149][150]152,.…”

Advancing Stem Cell Technologies and Applications: A Special Collection from the PluriCore Network in the German Stem Cell Network (GSCN)

“…This special collection brings together three articles on cellular differentiation and complex tissue models. Miller and colleagues report on the derivation of cardiomyocytes from pluripotent stem cells (Miller, Genehr, Telugu, Kurths, & Diecke, 2020), and Döpper and colleagues describe generation of retinal organoids that contain all seven retinal cell types (Döpper et al., 2020). Next, Applet‐Menzel and coworkers review human iPSC−derived blood‐brain barrier models as tools for preclinical drug discovery and development (Appelt‐Menzel et al., 2020).…”