**Abstract**

Retinal organoids (ROs) are 3D tissue structures derived from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) in vitro, which characterize the structure and function of retina to a certain extent. Since 2011, mouse and human retinal organoids have been available, opening up new avenues for retinal development, disease and regeneration research. Over the decade, great progress has been made in the development of retinal organoids, which is reflected in the improvement of differentiation efficiency and development degree. At the same time, retinal organoids also show broad application prospects, which are widely used in the construction of disease models. On this basis, the mechanism of disease, drug screening and retinal regeneration therapy have been explored. Although retinal organoids have a bright future, the deficiency of their structure and function, the limitations of differentiation and culture, and the difference compared with embryonic retina still remain to be solved.

**Keywords:** retinal organoids, retinal differentiation, disease models, retinal degenerative diseases, transplant

## **1. Introduction**

Located in the back of eyeball, the retina is a soft and transparent membrane attached to the inner surface of the choroid and forms part of the central nervous system. The retina can sense light stimuli, convert the light signals it receives into electrical signals, and then transmit them to the cerebral cortex through the optic nerve to form vision [1]. The retina is mainly composed of pigment epithelial cells, photoreceptor cells, bipolar cells, horizontal cells, amacrine cells, ganglion cells, and Müller glial cells [2]. Different neuron types form different layers, and the orderly arranged nuclei and synaptic regions are alternately arranged, forming a complex and orderly layered structure of the retina [3].

Our early research on the retina, derived only from human fetal retinal tissue [4, 5], faced significant challenges due to access difficulties and ethical issues [6]. Beyond that, most of what we know about the retina comes from studying animal retinas, but human and animal retinas differ in composition and function. For example, most mammals have only two types of cone photoreceptors that express S-opsin or M-opsin [7, 8], while humans have a third type that expresses L-opsin [9]; mice, the main subjects of our study, have a higher proportion of rods than humans [10], whose vision is determined by the density of cones in the macula and fovea [11, 12]. Therefore, it is of great significance to develop appropriate human retinal models to supplement animal models.

The establishment of human embryonic stem cell (ESC) lines [13] and the emergence of induced pluripotent stem cell (iPSC) technology [14] have turned our attention to cell research. Early 2D differentiation protocols used exogenous signaling molecules, Wnt antagonist DKK1 and bone morphogenetic protein (BMP) antagonist Noggin, to guide pluripotent stem cells to an anterior neural fate [15–17] and to differentiate into various types of retinal cells, including retinal pigment epithelial (RPE) cells, photoreceptors, and ganglion cells [18–25]. However, 2D differentiation is far from interpreting retinal development in vivo. Retinal development and maturation are regulated by a series of interacting signal networks, such as factors secreted by RPE that promote photoreceptor maturation. Early retinal differentiation produced a single cell type [19–22] and lacked the necessary interaction between cells. Therefore, we still need to find a more perfect model of human retina.

This breakthrough was achieved by constructing a 3D differentiation procedure. Through 3D differentiation, we can obtain retinal organoids that are highly reducible to the development process and complex structure of the retina, which we vividly call it "retina in a dish." In this chapter, we review the development of retinal organoids and show their application in today's life science research.

## **2. Overview of retinal organoids**

In 2011, Sassi's team used mouse embryonic stem cells (mESCs) to construct the first true retinal organoid through a 3D differentiation procedure [26]. In the following year, human retinal organoids were created [27], which is of epoch-making significance, meaning that human research on retinal development and retinal diseases has entered a new stage, and retinal organoids also provide a new and most potential tool for the treatment of retinal degeneration diseases.

During neurogenesis in vertebrates, the development of the retina can be roughly divided into two stages, the appearance of the optic cup structure and the orderly differentiation of seven types of retinal cells. In the first stage, the forebrain splits to form two secondary brain vesicles: telencephalon and diencephalon. In the diencephalon, eye field region first bulges outward to form the optic vesicle, and the distal vesicle invaginates to form the double-layer optic cup, which further develops into the outer retinal pigment epithelium and the inner neural retina (NR) (**Figure 1a**) [28–32]. In the second stage, the inner pluripotent retinal progenitor cells (RPCs) sequentially differentiate into retinal ganglion cells (RGCs), cone photoreceptors, horizontal cells, amacrine cells, rod photoreceptors, bipolar cells and Müller glia cells (**Figure 1b**) [33]. The cone and rod are connected to the retinal pigment epithelium and together form the outer nuclear layer (ONL). After extending to the outer plexiform layer (OPL), they form synapses with bipolar cells and horizontal cells in the inner nuclear layer (INL). On the other side of the inner nuclear layer, bipolar cells, amacrine cells, and ganglion cells form the synaptic networks of the inner plexiform layer (IPL). Müller glial cells span the whole layer of the retina, from the retinal pigment epithelium to the ganglion cell layer (GCL) (**Figure 1c**) [34, 35].

Retinal development in vivo is regulated by a series of transcription factors, signal transduction factors and cell surface factors. In vitro, differentiation of retinal organoids is also a programmed process that mimics development in vivo by adding various signaling molecules in stages. First, stem cells proliferate and aggregate (**Figure 2A**), inducing the formation of embryoid body (EB) (**Figure 2B**) and neuroepithelium (**Figure 2C**), which appear as translucent bright rings under a microscope (**Figure 2D**). And then,

*Retinal Organoids over the Decade DOI: http://dx.doi.org/10.5772/intechopen.104258*

#### **Figure 1.**

*Overview of retina. (a) the first stage of retinal development: The formation of double—Layer optic cup structure. (b) the second stage of retinal development: Retinal progenitor cells (RPCs) differentiate into seven types of retinal cells. (c) Structure of the retina.*

#### **Figure 2.**

*The differentiation of retinal organoids. (A) Growing human embryonic stem cells (H9). (B) Embryoid bodies (EB) at day 9 of differentiation. (C) Neuroepithelium appear at day 12. (D) Neuroepithelium appear as translucent bright rings at day 12. (E) Optic vesicle/cup at day 21. (F) Neural retinal (NR) region at day 26. Scale bars: 1000 μm (A, D, and E); 400 μm (B and C); 200 μm (F). All photos are provided by Dr. Ze-Hua Xu.*

they develop into optic vesicles (**Figure 2E**), followed by neuroretinas (**Figure 2F**), which in turn differentiate into seven types of retinal cells. The sequence of retinal cell types is consistent with in vivo development [36]. After differentiation, the cells undergo spontaneous nuclear migration, forming pinnacles and finally arranged into layered structures, in which the ganglion cells are located in the inner layer of the retinal organoid and the photoreceptors are located in the outer layer of the retinal organoid [26, 27]. Since RPE is usually a mass of cells not adjacent to the neuroretina and is not derived from the floating culture of optic vesicles, we do not consider it to be part of the retinoid organoid in this paper. With the continuous development of differentiation technology, photoreceptors in organoids become more and more mature, which is manifested by the appearance of outer segments and photosensitivity [37, 38].
