**2. Organoids in neural development**

CNS is generally regarded as the most complex system in human body. Limited by accessibility of living neural tissues and ethical challenges, human-specific features of neurodevelopment and neurological diseases remain largely unknown to us. Recent advances in stem cell technologies and 3D culture neural organoids have opened a new avenue in exploring the mechanisms of neurodevelopment. Early versions of the neural organoids range from complex neural epithelial structures to disorganized brain regions with large cellular diversity [5]. By supplementing exogenous factors and assembly of organoids during embryonic brain development, efforts have been made to gain the well-developed multilayer neural organoids and higher-order functions in terms of controlling patterning, morphogenesis, and function [6, 7].

#### **2.1 Neural organoids in brain development**

Through the embryonic brain development, neural progenitors progressively follow precise orchestration and coordination to acquire their spatial identities, a process characterized by successive changes in cellular composition and cytoarchitecture (**Figure 1a**). Dysregulation of this process may affect neurogenesis, synaptogenesis, and myelination and induce neurological or psychiatric disorders. To better investigate the early formation and function of the human brain *in vitro*, two different methodologies have been applied to generate human brain organoids: unguided and guided methods. The unguided methods rely largely on the capacity of spontaneous morphogenesis and intrinsic differentiation of the 3D aggregates while the guided organoid methods highly require supplementation of exogenous factors to induce hPSCs to differentiate toward specific brain regions [5]. Over the past decade, guided methods were induced by a set of growth factors and small molecules to induce the production of brain organs containing broad and specific identity, including forebrain, large cortical organoids, cerebellum, midbrain, and hippocampus. For instance, glycogen synthase kinase-3 (GSK-3) inhibitor, SMAD inhibitors, and WNT3A were for forebrain organoids induction [6]; SMAD inhibitors, Wnt activator, sonic hedgehog (SHH), and FGF8 were used for midbrain organoids induction [8]; FGF19 and SDF1 for cerebellum organoids induction [9]; WNT-3A and SHH were

*Applications of Neural Organoids in Neurodevelopment and Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.104044*

#### **Figure 1.**

*Schematic depiction of brain and eye development in vivo and in vitro. a. The timeline of brain development in human body and the features of human retinal development. b. Generation of hPSC-derived human brain organoids and retinal organoids in vitro, and the application of the generated organoids in regenerative medicine.*

used for hypothalamic organoids induction [10]. These generated brain organoids show robust neuronal and glial subtypes resembling the organization, transcriptional frameworks, and developmental timing of a primitive human fetal brain.

The 3D cultured brain organoids have been proven useful for many applications in basic research, for example, the development of the human brain cortex. It firstly begins with the expansion of the neuroepithelium, and then folds into six different layers. The main principles of the cortical layer formation are similar between mammals, such as primates and humans; however, the morphological differences are unneglectable. It is well known that neuronal number in primates massively increases in cortical surface, which eventually leads to the gyrification of the cortex (the generation of gyri and sulci) [11]. However, the mechanisms controlling the generation of gyrification are still not clear during the formation of cortical areas. The application of cortical organoids has helped us better understand the rapid expansion of human neocortex and the formation of cerebral cortical sulcus and gyrus. Karzbrun et al. revealed two opposing mechanical forces with the usage of

the brain organoids-on-a-chip: the middle cytoskeletal contraction and peripheral cell-cycle-dependent nuclear expansion, physically leading to differential growth and folding into brain wrinkling [12], and the extracellular matrix (ECM) components are implicated in neocortical expansion [13].

For another example, brain organoids are used to investigate the development of cellular interactions in the human brain. The human CNS originated from several distinct vesicles and then after a range of progenitors migrate and integrate, it moves into areas to generate multiple intertwined regions, ultimately resulting in emerging complex networks, neurons branching, and projecting. To model the intricate cellular interactions in human brain, fusing regionalized organoids into assembloids recapitulates more elaborate biological processes of brain development. This approach has been applied to study forebrain axis establishment, interneuron migration, oligodendrogenesis, and neuronal projections like the fused dorsal-ventral cerebral organoids to model interneuron migration in [7, 14, 15].

#### **2.2 Neural organoids in retinal development**

The eye originates from the ventral diencephalon, where a group of eye field transcription factors (EFTFs) are highly expressed such as PAX6, RAX, SIX3, and OTX2, and becomes specified as the eye field [16]. The eye field region is firstly split into optic vesicles in pairs and subsequently forms the optic cup by experiencing the valgus and invagination of the optic vesicle. The outer layer of the optic cup develops into retinal pigment epithelium(RPE) while the inner layer develops into neural retina. The neural retina with multilayered structure undergoes different phases of development, with different types of cells differentiating and maturing over the time (**Figure 1a**).

However, the understandings of the function and development of the human retinal are limited by scarce human fetal retina sample and species differences between animals and human. Since 2011, Sasai et al. released a landmark study to generate a self-organized 3D optic cup with layered neuroepithelia from mouse pluripotent stem cells (mPSCs), which opened a window for generating retinal models [17]. Many research groups have subsequently optimized the protocol to generate human retinal organoids derived from hPSCs. During retinal organoid generation, stem cell patterns the eye field-like regions expressing a complete component of the EFTFs to mimic the optic vesicle in early development [18]. What is encouraging, these tiny retinal organoids even contain almost all relevant retinal cell types: retinal ganglion cells (RGCs), amacrine cells, horizontal cells, bipolar cells, Müller cells, and photoreceptors.

#### *2.2.1 Retinal organoids in RGC development*

RGCs, the early-born neurons, transmit visual information between the eye and the brain, playing a critical role in retinal neuronal outputs. The loss of RGCs trends to result in a group of degenerative diseases such as glaucoma and optic nerve hypoplasia. Due to the specific time point of the RGC development, it is a challenge to obtain human fetus samples. In addition, the long-distance projection of neurites is the mostly important characteristic for RGC development as the extension of axons is regulated by extrinsic factors, including the ECM, growth factors, and glial cells. Recent several approaches have improved the capacity to differentiate hPSC-derived retinal organoids into RGCs that possess appropriate morphological and functional properties [19]. For example, Fligor et al. found that substrate composition including

### *Applications of Neural Organoids in Neurodevelopment and Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.104044*

laminin and Matrigel shows the most conducive for RGC neurite outgrowth; similarly, the growth factors with Netrin-1 and BDNF have the ability to guide and direct RGC axons outgrowth [20]. Besides, single-cell RNA sequencing (RNA-seq) results proved that the ganglion cells of retinal organoids at day 60 give the similar clusters to the human fetal retina on day 59 [21]. Collectively, the established retinal organoids serve as effective models for investigations of RGC development and disease modeling and as a valuable tool for cell replacement.

### *2.2.2 Retinal organoids in photoreceptors development*

Rod and cone photoreceptors are specialized neurons with functioning in the initial step of vision, which convert light stimuli into neurological responses. Rods are highly sensitive to light and operate under dim lighting conditions while the cones control color vision and high visual acuity. It is reported that the progressive loss of photoreceptors leads to blindness-associated inherited retinal diseases(IRDs), such as well-known retinitis pigmentosa (RP), congenital stationary night blindness(CSNB), and Leber congenital amaurosis (LCA). Therefore, it is particularly important to understand retinal progenitor fate choices toward rod photoreceptors and cone subtypes during retinogenesis. As such, the phototransduction mechanism requires a complicated cascade of gene regulatory networks. Aiming to induce hPCS-derived retinal organoids with mature photoreceptors, efforts of genetic manipulation and transcriptomic analysis have become the focal point for researchers [22]. Most recently, NRL (neural retina leucine zipper)-/- human-based 3D organoids were used to uncover the regulative role of MEF2C in cones' development [22]. RNAseq analysis of hPCS-derived retinal organoids has identified certain molecular signatures related with human photoreceptors development [23]. In brief, these observations and datasets have enabled to reconstruct developmental trajectories and recapitulate dynamics *in vivo* photoreceptors development.
