**3.1 Diversity of differentiation methods**

There are various differentiation methods for retinal organoids, but in terms of differentiation steps, there are mainly two differentiation schemes (**Figure 4**). The first is a classic 3D differentiation protocol from Sassi's team [26, 27]. The stem cells were dissociated and reassembled in a serum-free and low-growth factor medium (SFEBq culture, or serum-free culture of embryoid body-like aggregates with quick aggregation), and forced to form an embryoid body (EB) in a 96-well V-shaped plate.

**Figure 3.**

*Progress in retinal organoid differentiation over the decade.*

### **Figure 4.**

*Two main methods of retinal organoid differentiation.*

They were then stimulated by the addition of Matrigel to differentiate into neuroepithelial cells and subsequently into retinal progenitor cells and double-layer optic cup structures [27]. The cells were in suspension culture during the whole process of differentiation, and the formation of optic cups and the differentiation of neuroretina were self-organized [27]. 3D differentiation protocol is complicated in the early stage of differentiation, but it has a higher degree of reduction in the retinal development process, including the occurrence of optic cups invagination, the appearance of ciliary marginal stem cells at the NR-RPE boundary [39], and the establishment of dorsal-ventral (D-V) polarity [40].

The second differentiation method combines 2D culture and 3D culture (2D/3D) [37, 41–46], and the difference is mainly reflected in the early stage of neural induction. It has been reported that pluripotent stem cells can differentiate into the retina even when they are simply fused together [41, 42]. In this differentiation scheme, the stem cells were divided into small pieces by enzymatic hydrolysis [37] or mechanical methods [41, 43, 45] to form aggregates. The aggregates were cultured on a plate coated with Matrigel or floated in medium in the form of lumps of Matrigel/ PSCs [43, 45]. After it differentiated into neuroepithelium and optic vesicles, the latter were separated for suspension culture and further differentiated into retinal organoids. This approach bypasses EB formation stage and induces optic vesicle formation by endogenous production of inducer molecules from aggregated cells, avoiding the aggregation step of SFEBq method and the need of Wnt/BMP antagonist [47]. These studies suggest that cell-cell and cell-extracellular matrix interactions are key to inducing retinal organoids differentiation in the early stage of stem cell differentiation.

With the improvement of differentiation methods, the structure of retinal organoids has been improved. Photoreceptors can reach advanced maturity, characterized by the formation of the inner and outer segments and connecting cilia of photoreceptors, the appearance of photosensitivity [37, 44], the expression of photoreceptor neurotransmitters, and the formation of synaptic bands [38, 44]. By adjusting the differentiation method, we can also change the proportion of cells in organoids, such as retinal organoids rich in cones or RGCs [45, 46], which is good for cell transplantation. Oxygen is also an important factor in regulating the differentiation of retinal organoids, and hypoxic conditions (5%) effectively produce vesicles and cups as well as more mature neuroretinas [48]. Another study showed that high oxygen (40%) promoted the formation of NR in EB, as well as the generation, migration and maturation of retinal ganglion cells during metaphase differentiation [49]. The co-culture of RPE with retinal organoids promoted the differentiation of photoreceptors [50], while the co-culture with brain organoids promoted the axon extension of RGCs [51]. More encouragingly, researchers have differentiated human brain organoids with bilaterally symmetric vesicles [52].

## **3.2 Modulation of signaling molecules**

Retinal development requires the regulation of a series of signaling molecules. Similarly, by adding different signaling molecules, retinal organoids differentiation can be regulated in vitro. Dickkopf-related protein 1 (DKK-1), a Wnt signaling pathway antagonist, salvages the self-organizing ability of stem cells to differentiate into retinal progenitor cells [53]. Insulin-like growth factor 1 (IGF-1) regulates the formation of retinal organoids and promotes the formation of the correct retinal lamellar structure by various retinal cells [54, 55]. In the absence of IGF-1, retinal lamination

was absent at the early stage of differentiation, while photoreceptors decreased and retinal ganglion cells increased at the late stage of differentiation [55]. Addition of docosahexaenoic acid and fibroblast growth factor 1 can specifically promote the maturation of photoreceptors including cones [56]. Replacement of widely used alltrans retinoic acid with 9-cis-retinoic acid in culture medium promoted the expression of rod photoreceptors rhodopsin and the maturation of mitochondrial morphology [57, 58]. COCO protein can block BMP/TGFβ/Wnt signaling pathway, enhance photoreceptor precursors, and promote s-cone differentiation and inner segment protuberances formation [59, 60]. During retinal development, s-cone appear first, followed by L/M-cones. This time transition from the designation of the s-cone to the production of the L/M-cone is controlled by thyroid hormone (TH) signaling [61].

#### **3.3 Combination of organoid technology and tissue engineering technology**

There is also a lot of innovative research that combines retinal organoid technology with emerging materials technology. The use of bioreactors improved retinal stratification and increased the production of photoreceptors with cilia and new outer segments [62]. In static culture, the development of retinal organoids may be limited by oxygen and nutrient diffusion, and rotating-wall vessel (RWV) bioreactors can accelerate and improve the growth and differentiation of retinal organoids [63]. The spherical structure of retinal organoids limits its interaction with host RPE and the remaining neuroretinas during transplantation. In order to create a planar retinal organoid, a biodegradable scaffold was developed that mimics the extracellular matrix of neuroretinas [64]. Retina-on-a-chip is a new microphysiological model of the human retina that integrates seven different basic retinal cell types and provides vascular-like perfusion to retinal organoids [65]. Arrayed bottom-lined micropores composed of bionic hydrogels, facilitated rapid retinoid tissue formation from mESCs aggregates in an efficient and routine manner [66]. Automated microfluidic devices with significantly reduced shear stress can maintain the long-term survival of retinal organoids [67]. For details of some other differentiation improvements [68–71], please refer to **Figure 3**.

## **4. Applications of retinal organoids**

As a three-dimensional multicellular structure formed by self-organization in vitro, retinal organoids can reproduce the development process of retina in vivo to some extent, and can be used to summarize some structural and functional characteristics of human retina. Meanwhile, they are the most promising tools for retinal disease research (**Figure 5**).

#### **4.1 Retinal organoids as disease models**

The reprogramming technique enables iPSCs-derived retinal organoids to retain the patient's genetic characteristics, allowing us to study a variety of retinal diseases in detail in a dish. To date, retinitis pigmentosa (RP), Laber congenital amaurosis (LCA), retinoblastoma (RB) and some other retinal diseases (**Table 1**) have been reproduced in dishes using retinal organoid technology [47, 98].

RP is a relatively common hereditary retinal disorder characterized by night blindness and progressive loss of visual field [99]. LCA, the main disease leading to

#### **Figure 5.**

*Applications of retinal organoids.*



#### **Table 1.** *Retinal organoids as disease models.*

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

congenital blindness in infants, accounts for more than 5% of hereditary retinopathy, with complete loss of binocular cone and rod function within 1 year after birth [100]. Both diseases have been reported to be associated with multiple pathogenic genes. By differentiating different genetically-mutated stem cell lines into retinal organoids [101], we can observe their disease phenotypes in dishes, including photoreceptor degeneration, ciliary morphology disorder, and various functional impairment at molecular levels. Retinoblastoma is the most common intraocular malignancy in children [102]. The main cause of retinoblastoma is the loss of RB1 gene expression [103]. RB1 gene is a tumor suppressor gene, but the mechanism of RB1 deletion leading to retinal cancer is not clear, one of the key questions is the origin of RB cancer. By constructing RB models based on retinal organoids [104], we successfully observed tumorigenesis in retinal organoids and demonstrated that RB originates from ARR3 positive precursors of mature cones during development [90]. Other disease models, such as s-cone syndrome, rod-cone dystrophy, Macular telangiectasia type 2, microphthalmia, Stargardt disease, X-linked juvenile cleft retina, have also contributed to our understanding of retinal diseases.
