Brain Organoids: A Revolutionary Tool for Modeling Neurological Disorders and Development of Therapeutics

Abstract

Brain organoids are self-organized, three-dimensional (3D) aggregates derived from pluripotent stem cells that have cell types and cellular architectures resembling those of the developing human brain. The current understanding of human brain developmental processes and neurological disorders has advanced significantly with the introduction of this in vitro model. Brain organoids serve as a translational link between two-dimensional (2D) cultures and in vivo models which imitate the neural tube formation at the early and late stages and the differentiation of neuroepithelium with whole-brain regionalization. In addition, the generation of region-specific brain organoids made it possible to investigate the pathogenic and etiological aspects of acquired and inherited brain disease along with drug discovery and drug toxicity testing. In this review article, we first summarize an overview of the existing methods and platforms used for generating brain organoids and their limitations and then discuss the recent advancement in brain organoid technology. In addition, we discuss how brain organoids have been used to model aspects of neurodevelopmental and neurodegenerative diseases, including autism spectrum disorder (ASD), Rett syndrome, Zika virus-related microcephaly, Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD).

Introduction

Studying human brain development and disease pathology is complex because the brain possesses unique and exceptionally intricate developmental characteristics, which differ significantly from those of other organs (Kelava & Lancaster, 2016a). The complexity of the brain is not the sole obstacle to studying it. The inaccessibility of brain tissue and the absence of suitable human in vitro models also impede research on this organ. Many studies have investigated the principles underlying brain development, often by employing model species such as mice (Bakken et al., 2015; Belgard et al., 2011; Jang et al., 2022; Lein et al., 2007; Ling et al., 2009; Miller et al., 2013; Tole et al., 1997). However, these models are not entirely suitable due to inherent heterogenic differences between animal and human models (Jang et al., 2022). In addition, ethical concerns have limited the use of animal models in disease modeling and drug screening (Sreenivasamurthy et al., 2022). Therefore, there is an increasing demand for human in vitro models that can recapitulate the developmental process of the human brain accurately.

The ability to differentiate human pluripotent stem cells (PSCs) into specific cell types has revolutionized research on organ development and disease modeling (Di Lullo & Kriegstein, 2017). The formation of rosette structures from PSCs in 2D culture demonstrated features resembling the neural tube of the developing brain (Edri et al., 2015; Shi et al., 2012). These included a pseudostratified epithelium with apicobasal polarity, mimicking the properties of neuroepithelial cells and radial glial cells. Subsequent studies have shown that human PSCs can be differentiated into neural precursors even without serum, growth factors, or other inductive cues, highlighting the remarkable ability of human PSCs to acquire neural identity spontaneously (Ying et al., 2003). However, 2D cultures lack essential cell-cell and cell-matrix interactions, making it challenging to replicate specific physiological features (D. Zhang et al., 2014). More recently, researchers have employed human induced pluripotent stem cells (iPSCs) to create organoids, which are 3D multicellular aggregates that are self-organized, self-assembled, and capable of simulating one or more organ functions (Atamian et al., 2021). In vitro organoids can replicate essential aspects of in vivo brain organogenesis (Di Lullo & Kriegstein, 2017). In addition to neurons and neural progenitor cells (NPCs), brain organoids can also contain glial cells such as astrocytes and oligodendrocytes in later developmental stages (Lancaster et al., 2013).

By introducing an extracellular matrix to support tissue growth, 3D cell culture has facilitated advancements in the field of brain organoids. As a result, brain organoids can now reflect the cellular diversity, micro-architectural characteristics, and in vivo developmental path of the human brain (Jang et al., 2022). Recent advancements in single-cell transcriptomics have made it possible to enhance brain organoid protocols by identifying cell type-specific gene expression in organoids (Atamian et al., 2021). In addition, progress in genetic engineering techniques has enabled the genetic modification or alteration of stem cells, embryoid bodies (EBs), and mature organoids, ranging from small point mutations to the complete removal or assimilation of genes (Fischer et al., 2019). Consequently, with the advent of recent technological breakthroughs in genetic engineering, epigenetics, high-throughput single-cell transcriptomics, and genome editing, brain organoids created from human iPSCs can be utilized to study the development of the human brain and brain diseases (Qian et al., 2019).

Although various culture protocols for the differentiation of human iPSCs into brain organoids have been established successfully, there are still significant challenges, including complexity, throughput, and reproducibility (V. Velasco et al., 2020). Brain organoids exhibit batch-to-batch variation in terms of function and maturity, suffer from limited diffusion of nutrients and oxygen into the core, leading to necrosis, and have insufficient maturity as compared to in vivo counterparts (Di Lullo & Kriegstein, 2017; Kelava & Lancaster, 2016b; Pa, 2018).

Microfabricated devices and microscale cell printing technologies such as pillar/perfusion plates coupled with microarray 3D bioprinting have shown promise in overcoming some of the current limitations in the generation of organoids (S. Kang et al., 2023; Lekkala et al., 2023; Shrestha et al., 2021). These technologies allow for the arrayed manufacture of size-controlled cell culture that yields more homogeneous organoids, improving assay throughput, and lowering costs. In addition, microfluidic devices have been found to increase nutrient delivery and exchange in the core of organoids (V. Velasco et al., 2020). In this review, we will focus on the protocols developed so far for the generation of brain organoids, their limitations, recent advances in culture techniques to improve organoid functionality and maturity, and their applications for modeling various neurodevelopmental and neurodegenerative diseases.

Methods of generating brain organoids

In 2001, Zhang et al. (Zhang et al., 2001) developed the first human neural rosettes from embryonic stem cells (ESCs), demonstrating that clusters of PSCs could be spontaneously guided toward a neural lineage and plated on coated dishes to produce neuroepithelial (NE) cells that self-organized to form rosettes. The rosette structures exhibited epithelial characteristics with a surrounding apical lumen that reflects the properties of an embryonic neural tube (Zhang et al., 2001). Ying et al. (Ying et al., 2003) later demonstrated the differentiation of ESCs into neural precursors in the absence of serum, inductive signals, and growth factors. This method showed that the cells acquire autocrine signaling, allowing PSCs to differentiate into neural identities on their own (Ying et al., 2003). In 2005, Watanabe et al. (K. Watanabe et al., 2005) further improved the serum-free floating culture of embryoid-bodies (SFEB) by utilizing specific inductive signals to generate forebrain neural precursors from EBs. Dorsal forebrain progenitors could be generated by modifying these procedures, which were able to generate cortical neurons with a similar temporal pattern as seen in vivo (Gaspard et al., 2008). When these aggregates were given more time to differentiate, they developed larger rosettes; this method is called serum-free floating culture of embryoid-body-like aggregates with quick reaggregation (SFEBq) (Eiraku et al., 2008). In 2011, Eiraku et al. (Eiraku et al., 2011) achieved the first fully self-organized 3D brain structures by creating self-organizing optic cups from human PSCs, demonstrating intact tissue architecture. They combined a changed medium formulation to promote retinal identity with maintenance in floating culture rather than plating on coated dishes, improved upon the prior SFEB and SFEBq procedures (Eiraku et al., 2011). The tissues they developed recapitulated the developing retina with remarkable fidelity. This research offered the first proof that neural tissue kept in 3D floating culture is capable of self-organization and development of histologically precise tissue architecture (Eiraku et al., 2011). Since then, several approaches have been developed for the generation of brain organoids, which are broadly divided into guided and unguided methods (Figure 1).

Figure 1.

Methods of generating brain organoids

In an unguided method, iPSCs have been dissociated to form EBs, which are then embedded in Matrigel without the use of extrinsic factors, resulting in a whole brain or cerebral organoid containing various regions of the brain (Lancaster et al., 2013; Lancaster & Knoblich, 2014). Lancaster et al. (Lancaster et al., 2017) used poly(lactide-co-glycolide) copolymer fiber microfilament while forming EBs to increase the surface area for improving the reproducibility of neural induction and cortical development. With this method, cerebral organoids developed rapidly with neuronal identity in 8–10 days and distinct brain regions within 20–30 days (Lancaster et al., 2013). Further studies using single-cell transcriptome profiling have shown that cerebral organoids also contain retinal photosensitive cells, excitatory and inhibitory neurons, astrocytes, and oligodendrocyte precursor cells (OPCs) (Quadrato et al., 2017). The unguided method of cerebral organoids allowed continuous neuroepithelial expansion with distinct regions of the brain, which recapitulates the early developing brain and is mostly suited for studying early neurodevelopmental disorders (Lancaster et al., 2013). Nonetheless, the unguided method of differentiation possesses significant challenges for quantitative and systematic studies due to the presence of cell diversity and heterogeneity.

The guided method of differentiation uses small molecules and patterning factors to induce the formation of specific regions of the brain. In the fetal brain, the inhibition of the bone morphogenetic protein (BMP) signaling pathway results in the neuroectoderm formation, and the WNT and sonic hedgehog protein (SHH) signals determine the dorsoventral axis of the brain whereas fibroblast growth factor 8 (FGF8) determines the anteroposterior fate of brain development (J. Kim et al., 2021). In most of the differentiation protocols (Arenas et al., 2015; Chambers et al., 2009; Eiraku et al., 2008; Jo et al., 2016; S. Velasco et al., 2019; Xiang et al., 2017), neural induction is first initiated by inhibiting the SMAD signaling pathway that inhibits the formation of mesoderm and endoderm, which is then followed by the addition of fate-specifying molecules to promote differentiation into the specific lineages.

Forebrain organoid differentiation requires patterning factors such as WNT, BMP, and transforming growth factor beta (TGF-β) inhibitors (Eiraku et al., 2008; Kadoshima et al., 2013; Rosebrock et al., 2022). Rosebrock et al. compared the organoids generated with triple inhibitors with the organoids generated from the inhibition-free protocol and the protocol with dual SMAD (TGFβ and BMP) inhibitors as a control. Their gene expression analysis showed that organoids generated with triple inhibitors (triple-i) showed enriched cortical markers as compared to those with inhibitor-free and dual SMAD inhibitors. In addition, triple-i organoids showed upregulated cortex-specific genes whereas dual SMAD inhibition organoids showed upregulated mid-hindbrain specific genes. Another study showed that WNT and BMP signals are required for the in vivo formation of the cortical hem and choroid plexus (Hébert et al., 2003; Lee et al., 2000) whereas the inhibition of TGF-β induces neuroectoderm formation and produces cortical neurons (D. S. Kim et al., 2010). Forebrain organoids were also developed by the use of SMAD inhibitors (SB431542) and WNT inhibitors (IWR1e) (S. Velasco et al., 2019). The organoids expressed dorsal forebrain progenitor markers such as empty spiracles homeobox 1 (EMX1) and paired box 6 (PAX6) (S. Velasco et al., 2019). Jo et al. (Jo et al., 2016) generated midbrain organoids with the addition of SB431542, Noggin, CHIR99021, SHH, and FGF8. The resulting midbrain organoids showed the expression of orthodenticle homeobox 2 (OTX2), the transcription factor separating the midbrain and hindbrain, exhibited functional dopaminergic neurons, neuromelanin, and secreted dopamine. Dual-SMAD inhibitors (SB43152 and Noggin) and the Wnt pathway activator (CHIR99021) promote neuroectodermal fate, whereas the addition of sonic hedgehog SHH and FGF8 direct towards mesencephalic fate. In addition, SHH addition activates subpallial markers such as NKX2.1 (Arenas et al., 2015; Chambers et al., 2009; Eiraku et al., 2008).

On the other hand, cerebellum organoids can be generated by using TGF-β and BMP inhibitors, insulin, FGF2, FGF19, and stromal cell-derived factor 1 (SDF1) (Muguruma et al., 2015). The addition of FGF19 encourages the spontaneous formation of dorsoventrally polarized neural tube-like structures. In addition, the combination of SDF1 and FGF19 promotes the development of a continuous cerebellar plate neuroepithelium with a three-layer cytoarchitecture and a rhombic-lip-like structure at one end similar to the embryonic cerebellum (Muguruma et al., 2015).

Cortical organoids have been developed by the addition of Noggin and recombinant human Dkk-1 protein (rhDkk1) which showed the telencephalic development of the first trimester with an expression of PAX6, forkhead Box G1 (FOXG1), and GABAergic neuron (Mariani et al., 2015). Another group has developed cortical organoids by using SB431542, LDN193189, and XAV939 (Xiang et al., 2017). XAV939 is an inhibitor of the WNT signaling pathway whereas LDN193189 is an inhibitor of the BMP signaling pathway. The resulting cortical organoids showed a radial arrangement of glial fibrillary acidic protein (GFAP) in the ventricular zone during corticogenesis as seen in vivo (Xiang et al., 2017). SATB2, late-born neurons were separated from CTIP2, early-born neurons, indicating the features of deep and upper cortical layers. In addition, the presence of astrocytes indicates that astrogenesis was also observed in cortical organoids, which is an essential feature of corticogenesis.

Pellegrini et al. (Pellegrini et al., 2020) have developed choroid plexus organoids by adding BMP4, the dorsalising factor, in combination with CHIR99021, the WNT activator, which promoted the development of choroid plexus epithelial cells. Choroid plexus organoids were enriched with transthyretin (TTR)-positive regions, showed higher expression of specific markers of choroid plexus, chloride intracellular channel 6 (CLIC6), hydroxytryptamine receptor 2C (HTR2C) with other choroid plexus proteins such as phospholipid transfer protein (PLTP), plectin (PLEC), carbonic anhydrase 2 (CA2), apolipoprotein E (APOE), and insulin-like growth factor binding protein 7 (IGFBP7) (Pellegrini et al., 2020). Choroid plexus organoid at the later stage of development by 100 days produced more mature cerebrospinal fluid (CSF)-like fluid.

Despite the use of diverse approaches to create distinct repertoires of brain regional identities, they all face common technical challenges. For example, the organoid tissue lacks critical developmental and patterning signals that are required for development into a fully developed, mature organ. Even though brain organoids form distinct brain areas, the lack of body axis means that they do not arrange themselves in a manner that mimics in vivo development (Lancaster et al., 2013). The organoid generation process continues to experience batch-to-batch variability, in which the quality and produced brain areas in different batches of organoids vary significantly (Lancaster & Knoblich, 2014). In addition, the absence of vascularization in the in vitro culture is another limitation. The vascularization of the subventricular zone (SVZ) is crucial for late development because it creates a niche for neural progenitors and facilitates effective neural progenitor differentiation (Javaherian & Kriegstein, 2009; Lange et al., 2016). This deficiency in vascularization is likely one of the mechanisms impacting the scarcity of SVZ progenitors and may also partially explain the challenges researchers have faced in reproducing the correct cortical plate formation. Focusing efforts on delivering signaling molecules deep inside the tissue, either by changing the cell culture technique or engineering breakthroughs, is important to accurately replicate in vivo development, with all its inherent complexity. Furthermore, the center of the organoid is necrotic due to the nutrient/oxygen penetration issue, which may interfere with its normal physiology, development, and potential pathways for neural migration. To address these issues various approaches have been performed recently to overcome the diffusion limitation with enhancement in the maturity of organoids.

Advances in brain organoid generation

The development of brain organoids has been advanced through the application of various engineering platforms, such as spinning bioreactors, microfluidic devices, and cellular engineering. These approaches have helped to reduce structural heterogeneity and improve neuronal development and maturation.

a. Bioreactors to overcome diffusion limitation in brain organoids

One of the major challenges in generating brain organoids was the limited diffusion of nutrients and oxygen into the core, which leads to necrosis. Several types of bioreactors have been used to facilitate dynamic organoid culture and improve the survival of brain organoids by reducing cell death and necrotic zones (Figure 2a) (Goto-Silva et al., 2019; Lancaster & Knoblich, 2014; Qian et al., 2016, 2018; Saglam-Metiner et al., 2023; Silva et al., 2021). For example, cerebral organoids generated in a spinning bioreactor promoted the exchange of nutrients and oxygen, which improved the growth and development of organoids into defined regions of the brain (Lancaster & Knoblich, 2014). However, spinning bioreactors for large-scale suspension cultures have limited commercial applications due to their low throughput and high resource demand. To address this issue, Qian et al. developed miniature spinning bioreactors to generate brain region-specific organoids (Qian et al., 2018). They demonstrated that organoids generated in this bioreactor recapitulate key features at cellular, structural, and molecular levels of the developing brain with reduced medium volume. The culture of these organoids in miniature bioreactors could increase throughput and reproducibility, leading to the use of organoids for compound testing and disease modeling. Qian et al. (Qian et al., 2016) generated forebrain organoids in a cost-effective, miniature bioreactor for modeling Zika virus exposure to demonstrate cell death with reduced cell proliferation, resembling microcephaly.

Figure 2.

Advances in the generation of brain organoids. (a) Bioreactors for organoid culture, (b) Microfluidic devices for organoid culture, (c) Vascularized brain organoids, (d) Fusion of region-specific brain organoids, (e) Air-liquid interface culture of brain organoids.

Silva et al. (Silva et al., 2021) generated cerebral organoids using a vertical-wheel bioreactor, integrating a spacious vertical impeller alongside a U-shaped base to foster a uniform dispersion of shear forces within the bioreactor. This design facilitated gentle and consistent blending with a reduced agitation speed for uniform mixing and particle suspension (Croughan et al., 2016; Silva et al., 2021). Organoids generated in this platform exhibited enriched extracellular matrix to better mimic the neural microenvironment. In addition, they also demonstrated faster cellular commitment with efficient generation of cellular identity.

Saglam et al. (Saglam-Metiner et al., 2023) used a microgravity bioreactor employing a horizontally rotating cell culture system (RCCS) to create an environment with laminar flow and microgravity conditions for reducing shear stress and cellular damage and increasing mass transfer. This setup enabled the generation of cerebral organoids by ensuring consistent and uniform hydrodynamic forces. The RCCS bioreactor led to precise control over cell-cell interaction for long-term culture, enhancing reproducibility and harvestability (Saglam-Metiner et al., 2023). Brain organoids cultured in the RCCS bioreactor exhibited notable enhancements in neurogenesis and corticogenesis, resulting in a distinctive layered organization within brain organoids that distinguished them from shaker and spinner systems. These enhancements include increased cellular diversity, with abundant populations of neurons, glial cells, and endothelial cells, and the development of structural brain morphogenesis. In addition, the brain organoids showed improved development of functional neuronal identities, including glutamate-secreting glutamatergic neurons, GABAergic neurons, and hippocampal neurons, as well as enhanced synaptogenesis characterized by interactions between presynaptic and postsynaptic components. Thus, this study demonstrated enhanced organoid maturation at both the functional and molecular levels.

b. Microfluidic devices for enhanced brain organoid maturity

There are several challenges in creating a biomimetic environment conducive to the generation of brain organoids. Although inherently low throughput, microfluidic devices have been introduced to enhance the maturity of brain organoids and closely mimic early brain development, which are difficult to achieve with traditional static cell culture (Figure 2b). Brain organoids cultured in microfluidic devices, also known as brain organoid-on-chip, are advanced tissue models with physiological relevance (Castiglione et al., 2022). With this microfluidic technology, organoid culture can be conducted in a controlled environment that optimizes critical factors such as temperature, pH, nutrient and oxygen delivery, and waste removal (Gupta et al., 2016). In addition, incorporating sensors and actuators into microfluidic devices can enable precise monitoring and control (Yin et al., 2016). The advancement in this technology has enabled the precise replication of organoid morphology and physiology, offering microscale structures and parameters that mimic in vivo conditions (Bhatia & Ingber, 2014). Optimizing critical parameters, such as cell-cell and cell-extracellular matrix interactions, cell type composition, tissue architecture, nutrient exchange, and physical and electrical stimulation, could significantly reduce batch-to-batch variability and increase fidelity (Bhatia & Ingber, 2014).

Multiple studies have been published on the use of microfluidic devices to improve organoid maturity and physiological relevance (Castiglione et al., 2022; Cho et al., 2021; Karzbrun et al., 2018). For instance, Cho et al. (Cho et al., 2021) encapsulated brain organoids in hydrogel into individual chambers within the medium chamber, which were transferred to a 24-well plate on a digital rocker to compare the difference in maturity between the organoids generated in static and dynamic culture conditions. This study revealed that brain organoids placed in the microfluidic device exhibited a higher number of Ki67+ and Nestin+ cells in comparison to those cultured in the static condition in the traditional microtiter well plates. Dynamic culture enhanced neurogenesis, the development of cortical layers, and the electrophysiological function of human brain organoids derived from PSCs (Cho et al., 2021). Karzbrun et al. (Karzbrun et al., 2018) used microfabricated organoids-on-a-chip to explore the mechanisms of wrinkling and folding during brain development. This research is particularly relevant to understanding neurodevelopmental disorders, where wrinkling of the brain is observed, but the underlying mechanisms are poorly understood. Using fluorescence imaging, the researchers demonstrated how convolutions develop after the organoid reaches a particular cell density and nuclear strain. Their findings revealed that two opposing forces are responsible for the differential growth in the organoid that leads to surface wrinkling. Specifically, cytoskeleton contraction in the organoid core and nuclear expansion in the organoid perimeter drive this process.

c. Vascularized brain organoid for enhanced maturity

In vivo, a vascularized network plays a crucial role in facilitating the transportation of wastes, nutrients, and oxygen, as well as inductive biochemicals and a structural framework for growth (Grebenyuk & Ranga, 2019). Although organoids have been increasingly studied in academic labs, there are significant challenges that need to be addressed before using them broadly for clinical applications. One of the major limitations is the formation of the necrotic core due to diffusion limitations of nutrients and oxygen, resulting in premature differentiation in the absence of vascular supply (Grebenyuk & Ranga, 2019).

Vascularization is critical for recovering this aspect of development as fetal vascularization begins at the third gestation week (Grebenyuk & Ranga, 2019). The vascular network can be developed with the secretion of growth factors such as vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) by adjacent tissues (Grebenyuk & Ranga, 2019). Besides endothelial cells (ECs), other cells such as pericytes, immune cells, and smooth muscle cells also play a significant role in vascularization (Grebenyuk & Ranga, 2019). The development of a mature vascular system is crucial for maintaining the ability of organoids to grow during prolonged cultures and for better simulating the in vivo intricate processes occurring in vitro.

A variety of methods have been investigated to vascularize brain organoids. Initially, vascularized cerebral organoids were created by transplanting organoids into the brains of immunodeficient mice (Mansour et al., 2018). They demonstrated progressive maturation of neurons, gliogenesis, synaptogenesis, and neuronal differentiation in transplanted cerebral organoids. Pham et al. (Pham et al., 2018) also generated vascularized brain organoids by differentiating iPSCs of a patient into endothelial cells (ECs) and brain organoids separately and re-embedding the brain organoids with ECs on day 34 (Figure 2c). The organoids were grown in vitro for 3–5 weeks and transplanted in immunodeficient mice. ECs, cluster of differentiation 31 (CD31)-positive blood vessels were observed in between neural rosettes and within the organoids after transplantation (Pham et al., 2018). Furthermore, the addition of vascular endothelial growth factor (VEGF) at a concentration of 50 ng/mL enhanced the formation of ECs without reducing the neuronal marker in EBs (Ham et al., 2020) (Figure 2c). VEGF was included in the concentration of 25 ng/mL in cerebral organoid differentiation media along with Wnt7a after 2 months of culture, which promoted vascularization in cerebral organoids (Ham et al., 2020). VEGF enhances the differentiation of ECs whereas Wnt7A regulates pericyte mesenchymal differentiation and brain angiogenesis (Ham et al., 2020). The organoids showed the presence of endothelial marker CD31 along with the tight junction marker claudin-5. On further treatment with Wnt7a and VEGF, the development of a pericyte-like outer lining was induced that encircled the vascular tubes. They confirmed the expression of pericyte marker α-smooth muscle actin (SMA) in the outer cells and CD31 and von Willebrand factor (VWF) in the inner cell using immunofluorescence staining, H&E staining, and differential interference contrast (DIC) microscopy (Ham et al., 2020). More recently, human embryonic stem cells (hESCs) were engineered to express human ETS variant 2 (ETV2) and were mixed with non-infected hESCs to generate human cortical organoids (hCOs) to form a complex vascular-like network in organoids (Figure 2c). ETV2 is primarily responsible for the development of ECs. The expression of tight junctions, nutrient transporters, and trans-endothelial electrical resistance (TEER) all increased in vascularized hCOs (vhCOs), which also acquired numerous blood-brain barrier (BBB) characteristics (Cho et al., 2021). In addition, Salmon et al. (Salmon et al., 2022) generated neurovascular organoids in a microfluidic chip by co-culturing the ECs and pericytes derived from human PSCs with cerebral organoids derived from human PSCs. Vascular cells in interaction with cerebral organoids formed neurovascular organoids within the chip. This approach generated a vascular-like system without inhibiting brain morphogenesis and supporting organoid maturation. They observed higher level of the mature neuronal nuclei marker (NEUN) at day 15 and day 30 in the co-culture system, demonstrating that cerebral organoids were rapidly matured in presence of vascular cells in comparison to mono-cultured organoids. Vascular sprouts were directed towards the organoids without being overlapped with each other, allowing a permissive environment for the co-differentiation of different cell types such as CD31+, ECs, βIII tubulin+ post-mitotic neurons, and platelet-derived growth factor receptor beta (PDGRFβ+) pericytes on day 15 (Salmon et al., 2022).

d. Assembling brain organoids (i.e., assembloids) for enhanced function

While cerebral organoid differentiation methods can generate tissues that resemble different, interconnected brain areas, the proportions and spatial organization of these areas are highly unpredictable (Pasca, 2019). Several research groups simultaneously devised novel methods to enhance the modeling of inter-regional interactions (Bagley et al., 2017; Birey et al., 2017; Xiang et al., 2017, 2019). At first, they separated human PSCs into discrete organoids specific to each brain area and combined them to create organoids with multiple distinct regional identities in a controlled model (Xiang et al., 2019). For example, Grebenyuk and Ranga (Grebenyuk & Ranga, 2019) showed the migration of fluorescence-labeled inhibitory neurons from the subpallium to the pallium-like part in assembloids, in which organoids resembling subpallium and pallium were generated separately and fused later to study the neuronal migration (Figure 2d). The spatial and temporal signals determine the dorsal-ventral identity during forebrain development. SHH is crucial for ventral patterning. The neuroectoderm generated by the use of dual SMAD inhibition was exposed to SHH and its agonist purmorphamine to generate ventral forebrain organoids (Bagley et al., 2017; Birey et al., 2017). The ventral forebrain consists of GABAergic interneuron, whereas the dorsal pallium consists of excitatory neurons thus the fusion of dorsal and ventral forebrain organoids allowed the study of the migration of interneurons into the cortex (Bagley et al., 2017; Birey et al., 2017; H. Kim, Xu, et al., 2019). Xiang et al. (Xiang et al., 2017) also developed medial ganglionic eminence (MGE) specific brain organoids by fusing cortical and MGE organoids. The migration of cortical and glutamatergic neurons from MGE organoids towards the cortical organoids consisting of GABAergic neurons was studied via viral labeling of neuronal cells (Xiang et al., 2017).

Another study combined thalamic and cortical organoids, to simulate the development of reciprocal thalamocortical axon projections, which is crucial for sensory-motor processing and attention (Xiang et al., 2019). This approach can be used to study dopaminergic and glutamatergic input to basal ganglia or the spinal cord by developing striatum and midbrain organoids and also can be combined with the thalamus to study cortico-thalamic interaction, showing the cortical circuit development by their interaction (Pasca, 2019). The function of glial cells in synapse formation and its contribution to complex behavior and higher-order cognition can be studied with multilineage assembloids (Pasca, 2019). Hypocretin-producing neurons in hypothalamic organoids with glial and neural cells obtained from patients with narcolepsy can be combined with immune cells from the same patient to study the cellular events resulting in the loss of cells. There are various other applications for fusing these specific regions of organoids. For example, tumor organoids can be fused with brain organoids to study tumorigenesis, or organoids can be used to study disease pathology.

e. Air-liquid interface culture for enhanced brain organoid maturity

Cerebral organoids may lack mature neurons in later stages of development due to limitations in nutrient and oxygen diffusion, resulting in premature development (Giandomenico et al., 2019). However, this can be overcome by transplanting the organoids into a rodent to generate vascularization, allowing for proper nutrient and oxygen supply, and promoting later neuronal maturation (Giandomenico et al., 2019). Transplantation of cerebral organoids has been shown to improve cell survival and promote neural connectivity with the host brain. This transplantation approach has demonstrated that cerebral organoids have the intrinsic potential to form functional connections, as long as their survival is maintained over the long term. However, this transplantation can only be carried out by skilled personnel and is tedious, time-consuming, and expensive. Thus, there is a need for an in vitro system that could retain scalability and overcome these limitations by efficiently supplying nutrients/oxygen to improve the maturity of organoids. To increase surface diffusion into the interior of the organoid, a sliced organoid culture at the air-liquid interface (ALI) was developed, which requires cutting the organoid into disks (Figure 2e). Giandomenico et al. (Giandomenico et al., 2021) cultured slices of cerebral organoids (CO) at the air-liquid interface and demonstrated the improvement of overall maturity as compared with whole organoids with increased cortical neurons and axon outgrowth. When compared to entire organoids, ALI-CO cultures may be kept alive for up to 1 year while displaying better morphology and survival (Giandomenico et al., 2019). Hypoxia assay revealed air-liquid interface slice culture technique lessens organoid cell death and permits the development of the cortical architecture (Qian et al., 2020). Slice culture with improved maturation and long-term culture may be helpful in replicating late human neurodevelopment, a time that is crucial for understanding adult-onset diseases like amyotrophic lateral sclerosis overlapping with frontotemporal dementia (ALS/FTD) (Szebényi et al., 2021).

Brain organoids for disease modeling

Brain organoids are suitable to represent neurodevelopmental disorders with real pathogenesis because they recapitulate the development of the fetal brain, mimicking different aspects in terms of the tissue structure and cellular composition (Sidhaye & Knoblich, 2021). Some of the neurodevelopmental disorders related to progenitor cell regulatory defects are premature differentiation, decreased proliferation, and cell cycle disruption that may be accurately examined in brain organoids (Qian et al., 2019). Autism spectrum disorder, Rett syndrome, and Zika virus-related microcephaly are some of the neurodevelopmental disorders discussed in this manuscript (Figure 3). Neurodegenerative diseases can also be studied using brain organoids with limited success because they are late onset and age-related. Neurodegenerative disease-relevant phenotypes may not be completely reproduced with brain organoids which mimic embryonic brain development. Nonetheless, we discussed some of the neurodegenerative diseases in this manuscript, including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease (Figure 3). Overall, brain organoids have paved the way to model these disease conditions more accurately in comparison to prior models (Figure 4). Thus, brain organoids could be an excellent experimental model for brain disorders.

Figure 3.

Modeling of neurodevelopmental and neurodegenerative diseases in brain organoids

Figure 4:

The applications of brain organoids

A. Neurodevelopmental disorders

Neurodevelopmental disorders are a group of disorders that can cause abnormal brain function due to defects in the developing nervous system (Baldassari et al., 2020). It may affect memory, learning ability, emotion, and self-control (Baldassari et al., 2020). The development of the nervous system is influenced by both environmental and genetic factors, and any changes in the development pathway could lead to neurodevelopmental disorders affecting several regions of the brain (D’Souza & Karmiloff-Smith, 2017; Masini et al., 2020). The factors that cause neurodevelopmental disorders include nutritional or immune disorders, genetic diseases, toxic environmental factors, physical trauma, etc. (D’Souza & Karmiloff-Smith, 2017). Neurodevelopmental disorders include autism spectrum disorder (ASD), motor disorders, intellectual disability, developmental language disorder, neurogenetic disorder, traumatic brain injury, fetal alcohol spectrum disorders, etc. (Baldassari et al., 2020; D’Souza & Karmiloff-Smith, 2017). Among them, we will focus on ASD, Rett syndrome, and Zika virus-related microcephaly in this manuscript.

a. Autism spectrum disorder (ASD)

ASD is characterized by impairment in communication and social interaction with repeated and restrictive behaviors, difficulty in the use of language, lack of social and emotional relationships, apathy, etc. (Baldassari et al., 2020; Geschwind, 2011). Nearly 25 to 100 individuals per 10,000 people in the UK are estimated to be affected by autism (Baird et al., 2006). ASD can be categorized as syndromic and non-syndromic. The syndromic form of autism is characterized by a specific set of clinical features and its pathophysiological trajectories converged to a limited number of affected pathways (Aigner et al., 2014), whereas non-syndromic autism is diagnosed by structured interviews and is caused by specific genetic mutations, copy number variation, deletions, or disruptions (Maussion et al., 2019). The lack of a human disease model limits ASD research to recapitulate the human pathology features and represent the genetic heterogeneity (Villa et al., 2021). However, the recent advancement in iPSC technology has provided a better promising cellular source for disease modeling and drug discovery (Villa et al., 2021). iPSCs differentiation into brain organoids recapitulating the complex brain structure in terms of cellular composition and structure have been considered as a predictive system for studying autism pathogenesis and progression (H. Choi et al., 2017). The most common copy number variation (CNV) related to autism is the deletion or duplication of the 16p11.2 region, and the cortical organoids generated from skin fibroblast of patients with this CNV have been used to study ASD (Urresti et al., 2021). Synapse number, neuronal proliferation, and maturation along with the size of the organoid were affected by introducing CNV (Urresti et al., 2021). In addition, the pathway related to ion-channel activity, Wnt signaling, neuron migration, synaptic-related functions, and actin-related cytoskeleton were dysregulated by the CNV which was confirmed by transcriptomic and proteomic profiling. Cortical organoids derived from iPSCs of ASD patients showed increased production of inhibitory neurons due to overexpression of FOXG1 transcription factor, highlighting the importance of the FOXG1 gene in autism (Mariani et al., 2015). Overexpression of FOXG1 in organoids derived from idiopathic ASD iPSCs leads to abnormal cell fate and proliferation, shortened cell cycle length, and an unbalanced ratio of inhibitory and excitatory neurons (Mariani et al., 2015). Forebrain organoids were generated from iPSCs of ASD patients with a homozygous truncating mutation in contactin-associated protein 2 (CNTNAP2) to study autism (de Jong et al., 2021). CNTNAP2 encodes a cell adhesion molecule that is responsible for regulating the development and physiology of both the central and peripheral nervous systems (Saint-Martin et al., 2018). Chromodomain helicase binding DNA binding protein 8 (CHD8) is one of the commonly mutated genes identified through exome sequencing in autism, which affects the critical aspects of brain development such as neurogenesis, axonal guidance, and neuron differentiation (P. Wang et al., 2017). CHD8 has been knockout by clustered regularly interspersed short palindromic repeat-associated protein 9 (CRISPR/Cas9) technology in iPSCs to develop cerebral organoids and study the disease pathology in vitro (P. Wang et al., 2017). Dysregulation of neurogenesis-associated genes increased the production of GABAergic interneuron, and significant upregulation of transcription factor 4 (TCF4) expression was observed from RNA sequencing analysis in organoids developed from CHD8 knockout iPSCs (P. Wang et al., 2017). Brain organoids were also generated with a mutation in three known genes related to autism, including Histone-lysine N-methyltransferase SUV420H1 (a.k.a., KMT5B), AT-Rich Interaction Domain 1B (ARID1B), and CHD8 in multiple cell lines from different donors and found out that all the risk genes affected the same population of GABAergic neurons and excitatory projection neurons similarly by either accelerating or slowing down the neuronal development (Paulsen et al., 2022). SH3 And Multiple Ankyrin Repeat Domains 3 (SHANK-3) deficient single neural rosette-derived cortico-striatal organoids were developed to model autism which showed disrupted zinc finger protein (ZNF) and deficit in the expression level of protocadherins (Y. Wang et al., 2021). They also demonstrated elevated intrinsic excitability with a reduced number of excitatory synapses. The proper function of the synapse (i.e., the junction between neurons) is regulated by SHANK-3. Nonetheless, there are only a few studies of ASD conducted so far with brain organoids. Since the functional connectivity of the brain’s local and long-range networks is altered in autism, in vitro models such as brain organoids that recapitulate connection is crucial (H. Choi et al., 2017). In addition, evidence suggests that variations in genes and genetic alterations during the neurodevelopmental process influence the emergence of autism (Gaugler et al., 2014; Klei et al., 2012). It is crucial to construct models that accurately represent disease characteristics to study autism and to demonstrate neurodevelopmental conditions caused by genetic variation (Fatehullah et al., 2016). The use of the CRISPR/Cas9 technology for genome editing in brain organoids helps to explore mutations that cause autism as the initiation of the disorder is determined by genetic background. Thus, further study with autistic brain organoids would be necessary to investigate the role of autistic genes in ASD development and treatment, along with environmental factors such as developmental neurotoxic compounds.

b. Rett Syndrome

Rett syndrome (RTT) is primarily caused by mutations in Methyl-CpG-binding protein 2 (MECP2) which encodes multiple epigenetic regulators related to neuropsychiatric disorders (Mellios et al., 2018). MECP2 regulates neuronal development and functions as an activator and repressor when it binds to methylated CpG dinucleotides of target genes through its methyl-CpG binding domain (MBD) and recruits chromatin remodeling protein through transcriptional repressor domain (TRD) (Cheung et al., 2011; Mellios et al., 2018). RTT results in autism-like behavior, cardiac and respiratory abnormalities, seizures, and disturbance in motor coordination (Neul & Zoghbi, 2004). Common symptoms of Rett Syndrome include a smaller brain size, reduced neuron size, diminished dendritic arborization, and lower synaptic density (Neul & Zoghbi, 2004). The first study was performed by Mellios et al. (Mellios et al., 2018) where they showed miR-199 and miR-214 involved in alpha serine/threonine-protein kinase (AKT) and extracellular signal-regulated kinase (ERK) signaling pathways for neural differentiation and neurogenesis that were upregulated in brain organoids derived from Rett syndrome patients. It has been demonstrated that miRNAs control several stages of neurogenesis, including the proliferation of neural stem cells and the differentiation and maturation of neurons (Lang et al., 2012). Two female RTT patients’ fibroblasts carried a single nucleotide substitution (missense mutation, 316C>T) in the methyl-cytosine-binding domain of MECP2 (i.e., RTT Mut1) and another one which carried single nucleotide deletion (frameshift 705delG) in the transcriptional repression domain that resulted in premature termination of the MeCP2 transcript (i.e., RTT Mut2) were reprogrammed to generate neurons in monolayer culture and cerebral organoid. Microtubule-associated protein 2 (MAP2), early neuronal marker doublecortin (DCX) expression was significantly lower with reduced maturation and complexity of dendritic neurons in RTT neurons in 2D culture. In addition, the expression of early neuro progenitor marker PAX6 was significantly higher which showed impaired neurogenesis in RTT patient-derived cerebral organoids (Mellios et al., 2018). Moreover, RTT mutant organoids showed an increased ventricular area with decreased radial thickness in comparison to the isogenic wild-type organoids. Furthermore, the electroporated GFP+ cells in RTT wild-type organoids exhibited greater migration distance in comparison to the one with electroporated GFP+ cells in RTT mutant organoids. In another study, Gomes et al. (Gomes et al., 2020) generated brain organoids recapitulating both dorsal and ventral sub-regions of the forebrain from RTT patient-specific iPSCs. R255X MeCP2 dorsal organoids showed impaired electrophysiology and calcium signaling whereas the ventral organoids showed decreased DCX expression. On fusing these organoids, they revealed an impairment in interneuron migration, migrating with shorter distances in comparison to the control fused organoids (Gomes et al., 2020). Recent advancement showed that the MeCP2 knockout cortical organoid with a smaller diameter in comparison to the control, increased its diameter after 1 month of exposure to Nefiracetam, a GABAergic, glutamatergic, and cholinergic agonist, and PHA 543613, an α7-nAChR agonist with proven neuroprotective effects. Compound treatment improved the expression level of synaptic genes with the increase in the spiking population of the RTT organoids, confirmed by RNA sequencing and electrophysiological analysis, respectively (Trujillo et al., 2021).

c. Zika Virus-Related Microcephaly

A flavivirus called the Zika virus (ZIKV) was initially discovered in rhesus monkeys in Uganda in 1947. When an outbreak occurred in Central and South America in 2015, it attracted a lot of public attention because infections among pregnant women were connected to babies with unusually small heads, or microcephaly (Mlakar et al., 2016; Ventura et al., 2016). Quantitative analysis revealed that ZIKV strain affects neural progenitors, increases cell death, decreases neuronal cell layer volume, and reduces proliferation(Qian et al., 2016). Brain organoids have been used in analyzing the effect of ZIKV infection on brain structure and determining the pathogenic pathways of this infectious disease (H. I. Chen et al., 2019). ZIKV exposure showed preferential infection of NPCs in brain organoids, ultimately resulting in a significantly smaller organoid (Cugola et al., 2016; Qian et al., 2016, 2017). Neurons and astrocytes were also vulnerable to ZIKV infection when organoids were exposed at later time points (56 to 119 days) (Janssens et al., 2019; Qian et al., 2016). Additionally, these infected cortical organoids exhibit several traits linked to congenital Zika syndromes, such as thinning of the neuronal layer, disruption of apical surface adherent junctions, and enlargement of the ventricular lumen, providing concrete proof that exposure to the ZIKV during embryonic development causes neurological disorders(Qian et al., 2016). In addition, Dang et al. (Dang et al., 2016) infected cerebral organoids with ZIKV strain MR766 on day 10, and the growth of the organoid was monitored for 5 days. ZIKV activated Toll-like receptor 3 (TLR3) in cerebral organoids that trigger apoptosis and attenuate neurogenesis (Dang et al., 2016) with the reduction in organoid volume that represents a neurological disorder microcephaly. Another study found that tyrosine kinase receptor (AXL) was upregulated in microglia during ZIKV infection in 75-day-old brain organoids (Billaud et al., 2017), indicating that AXL may play a part in ZIKV infection. ZIKV-infected organoids when treated with drugs showed decreased apoptosis (M. Watanabe et al., 2017; Xu et al., 2016), reduction in virus replication (Sacramento et al., 2017; Zhou et al., 2017), and protection from infection (M. Watanabe et al., 2017).

B. Neurodegenerative diseases (NDD)

The progressive loss of neuron structure or function in the brain is the root cause of neurodegenerative diseases that might result from protein aggregation, aging, genetic mutation, and toxicity (Chang et al., 2020). Cell death may ultimately result from such neural injury. Amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD), multiple system atrophy (MSA), and prion disorders are a few examples of neurodegenerative diseases. In the research of NDDs, brain organoids could be used as a scale model of a patient’s brain for understanding disease mechanisms and creating therapeutics and regenerative therapies for NDDs. In this manuscript, we focus on the most representative NDDs including AD, PD, and HD.

a. Alzheimer’s disease (AD)

AD is a neurodegenerative disease characterized by a neuropathological hallmark of extracellular plaques made of amyloid-beta (Aβ) peptides and intracellular tangles made of hyperphosphorylated aggregates of the microtubule-associated protein tau, which are present throughout the cerebral cortex and sub-cortical regions (Braak & Braak, 1991). It affects 50 million people worldwide (Wray, 2021). The mechanisms underlying the pathology of AD are largely derived from a cohort of people with early-onset familial AD (fAD) (Raja et al., 2016). These cases have causative mutations largely in the Aβ-processing enzymes presenilin 1 and 2 (PSEN1, PSEN2), which are a component of the secretase complex, or in the amyloid precursor protein (APP) gene itself, which may have mutations or duplications. In typical 2D cultures, secreted Aβ diffuses throughout a substantial amount of medium due to a lack of interstitial compartment (Raja et al., 2016). Choi et al. (S. H. Choi et al., 2014) demonstrated a unique strategy for restricting Aβ diffusion and enabling quick aggregation in a human neural stem cell-derived 3D culture. They showed enhanced Aβ aggregation and hyperphosphorylated tau protein (P-tau) in 3D-differentiated human neural progenitor cells (hNPCs) with FAD mutations in β-APP and PSEN1. Inhibitors of β- and γ-secretase reduced the levels of toxic Aβ₄₂ and phosphorylated tau in the 3D hNPC model (S. H. Choi et al., 2014). Another group developed organoids from the iPSCs of AD patients with PSEN1 and APP overexpression which showed an increased expression of Aβ markers D54D2 and 4G8 with increased tauopathy (Raja et al., 2016). The antibodies 4G8 and D54D2 have been extensively utilized to identify both soluble and aggregated Aβ (immunoreactive against amino acid residues 17–24 of Aβ) and several amyloid isoforms (Aβ₃₇, Aβ₃₈, Aβ₃₉, Aβ₄₀, and Aβ₄₂), respectively (Raja et al., 2016). Time-dependent accumulation of Aβ from 60 days to 90 days in vitro was also demonstrated in an organoid generated from an APP duplication carrier (Raja et al., 2016). Several researchers have studied AD pathologies such as elevated Aβ levels, expression of neuro-inflammatory markers, extracellular matrix remodeling, and synaptic dysfunction, which were faithfully represented by these iPSCs-derived organoids from AD patients (Gonzalez et al., 2018; Yan et al., 2018). Similarly, Gonzalez et al. (Gonzalez et al., 2018) developed FAD cerebral organoids with patient iPSCs carrying PSEN1 mutation in which Aβ and P-tau depositions were observed. Several groups have demonstrated Aβ pathology in iPSC-derived organoids harboring FAD mutations which provide evidence of the development of this pathology with endogenous expression of AD-associated mutations (S. H. Choi et al., 2014; Raja et al., 2016; Wray, 2021). In another study, Kuehner et al. (Kuehner et al., 2021) showed reduced 5-hydroxymethylcytosine (5hmC) levels in FAD forebrain organoids containing PSEN1 and APP mutations along with upregulation of genes related to AD in comparison to control organoids. The development of the embryonic brain is correlated with the existence of 5hmC through neural differentiation, cell functions, and structural development (Sreenivasamurthy et al., 2022). The lack of 5hmc is the potential onset of FAD during fetal human brain development (Kuehner et al., 2021).

Sporadic Alzheimer’s Disease (SAD) has been deemed more challenging to predict, although it includes disease pathologies similar to those associated with FAD as Aβ accumulation, P-tau, Neurofibrillary tangles (NFTs) development, and loss of neurons due to the lack of related mutations and the impact of environmental stimuli (Sreenivasamurthy et al., 2022). The onset of SAD was strongly linked with the E4 variant of apolipoprotein E (APOE4) (Al Mamun et al., 2020). Lin et al. (Lin et al., 2018) showed that Aβ accumulation in organoids containing duplicate APP mutation when co-cultured with microglia-like cells carrying the APOE4 allele was more than APP-duplicated organoids co-cultured with microglia-like cells carrying the E3 variant of apolipoprotein E (APOE3 allele). The findings imply that APOE4 has a detrimental effect on several microglial functions that may impair microglia’s ability to remove extracellular Aβ from AD brains and may also change the brain’s inflammatory profile (Lin et al., 2018). Lower Aβ formation was seen in SAD patient-derived organoids that had undergone APOE4 to APOE3 editing (Zhao et al., 2020). Similar to this, Chen et al. (X. Chen et al., 2021) created a cortical organoid model to study blood-brain barrier (BBB) leakage frequently seen in SAD by exposing organoids to human serum for 12 days. As predicted, beta-secretase 1 (BACE), one of the proteins that cleave APP to produce Aβ aggregates, was expressed more often in organoids that had been exposed to serum (X. Chen et al., 2021). Thus, brain organoids hold promise for studying AD development and pathogenic processes, including gliosis, inflammation, synaptic plasticity, and lipid metabolism (Chang et al., 2020).

b. Parkinson’s disease (PD)

PD is characterized by the accumulation of Lewy Bodies consisting of insoluble aggregates of α-synuclein that leads to the movement disorder (Wray, 2021), affecting 2 in 1,000 individuals (Valkovic et al., 2015). Dopaminergic neurons of the substantia nigra are degenerated by this accumulation of Lewy Bodies, leading to postural instability, tremors, slowness in movement, and muscle rigidity (Valkovic et al., 2015). α-Synuclein protein-encoding gene SNCA and glycine to serine substitution G2019S, located within the kinase domain encoded by exon 41 of Leucine Rich Repeat Kinase (LRRK2), are commonly mutated genes in PD. Kim et al. (H. Kim, Park, et al., 2019) generated midbrain organoids with G2019S mutation in LRKK2 for modeling PD and observed the accumulation of α-synuclein protein, which was similar to the transcriptomic profile of the post-mortem brain tissue with similar mutation. The organoids showed abnormal localization of Ser-129 phosphorylated α-synuclein with increased levels of autophagy marker, light chain 3B (LC3B), like the one seen in LRRK2-associated PD patients. This patient-derived organoid model may be relevant for drug discovery because treatment with a specific LRRK2 kinase inhibitor (GSK2578215A) decreased the accumulation of phosphorylated-synuclein and neuronal cell death in the organoids (H. Kim, Park, et al., 2019). LRRK2 mutation resulted in an increased level of the thioredoxin-interacting protein (TXNIP) gene, in mouse and PD culture models (H. Kim, Park, et al., 2019) and increased levels of Forkhead box A2 (FOXA2) with a reduction in branching and complexity of dopaminergic neurons in LRRK2 G2019S midbrain organoids (Smits et al., 2019). An interaction between α-synuclein and LRRK2 was shown by the reduction of α-synuclein aggregation in LRRK G2019S mutant organoids when the TXNIP gene was knocked down (H. Kim, Park, et al., 2019). In addition, Chlebanowska et al. (Chlebanowska et al., 2020) generated midbrain organoids from iPSCs of PD patients which showed decreased levels of LIM homeobox transcription factor alpha (early) and tyrosine hydroxylase (late) markers between organoids from PD patients and healthy volunteers. LMX1 plays a key role in midbrain dopaminergic neuron development and its survival (Chlebanowska et al., 2020). The midbrain organoids derived from PD patient iPSC showed reduced differentiation efficiency to tyrosine hydroxylase positive (TH+) neurons with apoptosis, mitophagy, and imbalanced proliferation (Jarazo et al., 2022). Treatment with 2-hydroxypropyl-β-cyclodextrin improved the dopaminergic differentiation of neurons in midbrain organoids derived from PD patient iPSCs (Jarazo et al., 2022).

c. Huntington’s disease (HD)

HD is a fatal inherited NDD that results from the expansion of a repeating cytosine-adenine-guanine (CAG) triple series in exon 1 of the huntingtin gene (HTT) (Walker, 2007). It leads to progressive cognitive deterioration, behavioral changes, motor dysfunction, and lack of coordination (Walker, 2007). An excessive amount of CAG repeats in the brain and striatum leads to cell death, particularly in medium-spiny neurons. This results in numerous cellular changes as abnormalities in vesicular trafficking, mitochondrial and metabolic activity, gene transcription, and ATP levels. Using a specialized micro-device, Kawada et al. (Kawada et al., 2017) developed a motor nerve organoid from human iPSCs. The device contained a microchannel that supported axon growth while also facilitating their organization into a unidirectional fascicle, as demonstrated by the production of the presynaptic protein synaptophysin and the axonal protein Tau1. In addition, these organoids were positive for the motor neuron marker SMI32 (neurofilament H [non-phosphorylated]) and the general neuronal marker Tuj1 (Class III beta-tubulin) (Kawada et al., 2017). These methods can be applied to different types of neurons in the nervous system (e.g., corticospinal tract), corpus callosum, and sensory nerve to model axon fascicles (Kawada et al., 2017). In addition, neuron interaction through synaptic connections can be studied by providing postsynaptic target cells (Kawada et al., 2017). Thus, axon fascicles connection with neurons or non-neuronal target cells can be studied in vitro by using a nerve organoid model (Kawada et al., 2017). Conforti et al. (Conforti et al., 2018) showed abnormal neuronal maturation and disrupted cellular organization in cortical and striatum organoids derived from HD iPSCs and found out that mutant HTT (muHTT) has an impact on this effect. In the next step, a synthetic zinc finger protein (ZFP) repressor was used to downregulate muHTT which rescued the defect in neuroectodermal development. The hypothesis of muHTT impact in neurodevelopment and neurogenesis was further demonstrated by Walker et al. (Walker, 2007) in HD iPSC-derived brain organoids which showed reduced organoid size, disorganized neural progenitors, lower neuroepithelial structures, and increased neural cell cycle activity resulting from the negative influence of the protein kinase ataxia telangiectasia mutated (ATM). These motor neuron organoids may serve as a resource for understanding the mechanisms behind HD, and for exploring novel HD therapy.

Conclusion

Brain organoids have enabled the replication of in vivo phenomena in human tissue in a controlled microenvironment. The integration of transcriptomic and proteomic profiling has increased the utility of brain organoids in crucial aspects of neurodevelopment, pathogenesis, and disease progression. With longer differentiation and maturation time, brain organoids acquire more representative in vivo characteristics and functionality, making them suitable models for targeted therapeutics and a source of fully functional tissue for regenerative medicine applications. These organoids can also be used for high-throughput preclinical screening and subsequent validation. Despite the continuous adoption of organoid technology, further improvements are necessary to fully realize their potential for clinical applications. A highly accurate and reproducible culture model is expected to emerge when combined with more defined extracellular matrices (ECMs), standardized culture platforms, and validated organoid culture media. In addition, the incorporation of other cell types, including endothelial cells and immune cells, to enhance functional maturation will drive this field toward more comprehensive and accurate models. Thus, brain organoids are one of the most intriguing and promising technologies to have emerged recently and have the potential to be accessible to a wide range of clinical scientists and academics for maximizing their potential.

Table 1.

Methods of brain organoid generation

Differentiation	Organoid Type	Extrinsic factors	Platform used for culture	Ref.
Unguided Method	Whole brain organoid	-	Spinning bioreactor	(Lancaster et al., 2013)
	Whole brain organoid	-	Spinning bioreactor	(Lancaster & Knoblich, 2014)
Guided Method	Forebrain organoid	Dorsomorphin, A83–01, WNT-3A, CHIR99021, and SB431542	Spinning bioreactor	(Qian et al., 2016, 2018)
	Midbrain organoid	LDN-193189, SB-431542, SHH, Purmorphamine, and FGF-8
	Hypothalamus organoid	LDN-193189, SB-431542, WNT-3A, SHH, and Purmorphamine
	Forebrain organoid	IWR1e and SB431542	Spinner flask	(S. Velasco et al., 2019)
	Midbrain organoid	SB431542, Noggin, CHIR99021, SHH, and FGF8	Ultra-low attachment (ULA) 6-well plate on an orbital shaker	(Jo et al., 2016)
	Cerebellum organoid	SB431542, insulin, FGF2, FGF19, and SDF1	Petri dish	(Muguruma et al., 2015)
	Cortical organoid	Noggin and rhDkk1	Petri dish and ULA 96-well plate	(Mariani et al., 2015)
	Cortical organoid	SB431542, LDN193189, and XAV939	ULA 6-well plate on an orbital shaker	(Xiang et al., 2017)
	Choroid plexus organoid	BMP4 and CHIR99021	Petri dish	(Pellegrini et al., 2020)