Usage of Brain Organoids to Help Decode the Mechanism of Genetically Induced Microcephaly

Abstract

During the process of decoding the mechanism of microcephaly, limitations of animal models and cell lines made them not able to give out a clear solution. Moreover, the ethical problems have also pulled back the progress. But with the development of technology, organoids have become one important strategy in the research of microcephaly. Having the ability to model different cell interactions and their 3-D structure, organoids can not only reveal unknown aspects of neurogenesis but also the roles of different genes during brain development. In the case of gene-induced microcephaly, the use of cerebral organoids and patient-specific organoids has helped researchers to decode the mechanism of both primary and secondary. However, unsolved questions and challenges still appear in the research. Therefore, a discussion on the topic of the limitations of organoids will be given.

Keywords: organoid, patient-specific organoid, brain development, microcephaly, neurogenesis

Introduction

The human body can be viewed as a sum of a wide variety of cellular and non-cellular materials that formed in a highly organized manner such as cells, tissue, and organ), moreover, the entire interaction in the human body that includes internal (e.g. cell-cell, cell-matrix) or external (e.g. cell-environment) interactions are also essential to the composition and the overall movement of the human body¹.

Over time, scientists have used animal testing and cell lines to mimic this sophisticated structure to reveal mechanisms behind certain diseases. However, both animal testing and cell lines have many limitations. Human neural progenitor cells (NPCs) exhibit distinct characteristics compared to rodent NPCs, including a longer neurogenic period and a more intricate cerebral cortex structure—features poorly represented in rodent models. Crucial cell types like basal radial glial (bRG) cells, essential for human cortical expansion, are rare or nonexistent in rodents².³.  Moreover, human interneuron migration patterns and microglial immune functions—vital for understanding neuroinflammation in microcephaly—differ markedly between species⁴. While cell lines offer valuable insights, they lack the in vivo context and cellular interactions crucial for accurately modeling brain development. They also fail to replicate key structures such as three-dimensional tissue architecture⁵. Furthermore, the variability in human induced pluripotent stem cells(hiPSC)-derived cell lines, stemming from intrinsic epigenetic differences, hampers their reliability as models, often leading to unpredictable differentiation outcomes⁶ .

To study this complex system scientists had to overcome the ethical limitations of directly examining the human body by creating clinically relevant models. The hierarchical structure inherent in living organisms implies that multi-level recapitulation, which is the repetition of an evolutionary or other process during the development or growth, of the body could be achieved by using model systems with diverse cell types and their interactions, and organoids fit the demands by having 3-D structures grown from stem cells which exhibit remarkable self-organizing properties that result in the ability to reflect key structural and functional properties of organs⁷. Experimental animals are frequently used in laboratories despite the fact that it’s hard to reproduce animal models in that environment.

In create high throughput systems as well as increase reproducibility, experiments on cell lines offer useful alternatives in modeling certain functions of the human body. The middle ground between the two extremes was long missing when a radically new model system entered the research laboratories. The scientific world named it: organoid.

Organoids, different from cell lines, are tiny three-dimensional assemblies of cells that contain multiple cell types and exhibit some of the physiological characteristics of the organ to which the cells belong⁸.  Compared with cell lines, organoids address limitations such as the absence of interaction between different cell types that compose tissue and cell-matrix interactions necessary for maintaining defining in researching the phenotypes and mechanism of microcephaly, resulting in an inability to mimic tissue-specific cellular functions and signaling pathways⁷. Organoids are generated from either pluripotent stem cells or adult stem cells by mimicking biochemical and physical incentives of an organ’s development⁵. In the process of organoid formation, the organoid system is been governed by the stem cell microenvironment, which is composed of a wide range of biochemical and biophysical signals that control the cell-cell interactions and cell-matrix interactions⁹. Although current organoid systems still rely on instinct and external biochemical signals, such as growth factors, the formation of organoids is still dependent on cell-autonomous self-organization⁵.   

Furthermore, organoids recapitulate a large number of biological parameters including the spatial organization of heterogeneous tissue-specific cells, diverse cell-cell interactions, cell-matrix interactions, and certain physiological functions generated by tissue-specific cells within the organoids¹⁰.The pioneering study of cerebral organoids from Lancaster demonstrated that cerebral organoids can recapitulate many aspects of human brain development, including the formation of various brain regions and the outer radial glial stem cell population, which is involved in cortical expansion. Additionally, the study modeled microcephaly, showing how the organoids can be used to study brain development disorders¹¹. Moreover, the research of Takasato and colleagues demonstrated that kidney organoids derived from hiPSCs can recapitulate the complex structure and cell types of the developing kidney, including nephron structures and various kidney cell types. These organoids also exhibited functionality akin to human kidneys, such as filtration and reabsorption properties¹².

Organoids have played a crucial role in microcephaly research. Pioneer researchers like Lancaster and Gabriel used organoids developed from patient cells to understand the mechanism of microcephaly¹³,¹⁴.  Microcephaly is a disease marked by symptoms such as altered neural progenitor cell behavior, such as premature differentiation or increased apoptosis¹⁵. Unregulated genes and pathways, which co-evolved with the human brain, can result in a smaller brain, particularly a smaller frontal cortex. This condition is clinically known as primary microcephaly. Microcephaly is further divided into primary and secondary types. Primary microcephaly involves early brain development abnormalities, leading to disproportionate cortex thickness. Secondary microcephaly occurs after birth during infancy¹⁶. Several genes associated with microcephaly include MCPH1, AUST2, and CTNNB1¹⁷. To be specific, MCPH1 (Microcephaly Protein 1) is directly associated with primary microcephaly. It plays a crucial role in cell cycle regulation and the maintenance of neural progenitor cells. Mutations in MCPH1 can disrupt normal brain growth, leading to reduced head size; AUST2 is involved in the regulation of gene expression and mRNA stability. Abnormalities in this gene can affect neural differentiation and proliferation, contributing to microcephaly by impairing normal brain development; CNNB1 (Catenin Beta 1) encodes β-catenin, a key component of the Wnt signaling pathway, which is vital for neural stem cell proliferation and differentiation. Mutations or dysregulation of CTNNB1 can lead to developmental defects, including microcephaly, by affecting these critical processes.

Various types of organoids have been instrumental in brain development research, particularly in investigating the mechanisms underlying genetically induced diseases like microcephaly. These organoids, such as Expanding Neuroepithelium Organoids (ENOs) and Cerebral Organoids (COs), will be discussed later in this document¹⁸.

Gene	Syndromes	Mechanism
ASPM	congenital microcephaly	decreased NPCs proliferation and less neuronal activity
ATR	seckel syndrome	mitotic delay, impaired cytokinesis, double-strand repair deficiency
CDK5RAP2	congenital microcephaly	decreased NPCs proliferation,  premature NPCs differentiation
CDK6	congenital microcephaly	abnormal spindle + unknown mechanisms
CENPJ/CPAP	congenital microcephaly, seckel syndrome	decreased NPCs proliferation,  premature NPCs differentiation
CEP135	congenital microcephaly	abnormal centriole structures, disorganized spindles, reduced NPCs proliferation
CEP152	congenital microcephaly, seckel syndrome	decreased NPCs proliferation
CEP63	seckel syndrome	increased neuronal death, increased mitotic error
CIT	congenital microcephaly, dwarfism	mitotic delay, impaired cytokinesis, multipolar spindles, genomic instability, cell death
DNAPL	congenital microcephaly, seizures, neuronal death	Double-strand break repair deficiency
ERCC6, ERCC8	Cockayne syndrome, microcephaly	nucleotide excision repair and base excision repair deficiency
KIF11	congenital microcephaly	abnormal spindles and reduced NPCs proliferation
KIF14	congenital microcephaly, Meckel syndrome	increase neuronal cell death, abnormal cell migration
KIF2A	cortical dysplasia	abnormal axon branching, abnormal microtube function
KIF5C	cortical dysplasia	abnormal microtubule function
ligase IV deficiency	congenital microcephaly	double-strand break repair deficiency, neuronal death
MCPH1	congenital microcephaly	premature NPCs differentiation, chromosome condensation
NBS1	congenital microcephaly, Nijmegen breakage syndrome	double-strand break repair deficiency
NDE1	congenital microcephaly	decreased NPCs proliferation
Ninein	seckel syndrome	defective migration, neuroectoderm defects
PCNT	congenital microcephaly, seckel syndrome, MOPD type II	decreased NPCs proliferation, aberrant mitosis, missegregation of chromosomes
RTTIN	congenital microcephaly, dwarfism, cerebellar abnormalities	abnormal spindles, centriole structures
SAS6	congenital microcephaly	decreased NPCs proliferation
STIL	congenital microcephaly	neural tube defects
TTDA	congenital microcephaly	Double-strand break repair deficiency
TUBA1A	cortical abnormalities tubulopathy	abnormal neuronal migration
TUBB2B	cortical abnormalities tubulopathy	abnormal neuronal migration
TUBG1	cortical abnormalities tubulopathy	abnormal neuronal migration
WDR62	congenital microcephaly  cortical abnormalities	decreased NPCs proliferation,  premature NPCs differentiation
XLF/Cernunos	congenital microcephaly	double-strand break repair deficiency, neuronal death
XPA-XPG	xeroderma, pigmentosum, variable microcephaly	Double-strand break repair deficiency
XRCC2	congenital microcephaly	double-strand break repair deficiency, neuronal death
XRCC4	congenital microcephaly	double-strand break repair deficiency, neuronal death

ORANGANOID FOR MICROCEPHALY

1. Modelling the Human Brain

1.1 Experimental Model: Human-Mouse Chimera

The human brain is larger than the mouse brain, and also much more complex, with more neocortical invaginations and a greater number of specialized areas. However, at the level of individual neuron types and their connections, mouse and human brains are similar. Despite this general conservation, we also found extensive differences between homologous human and mouse cell types, including marked alterations in proportions, laminar distributions, gene expression, and morphology. These species-specific features emphasize the importance of directly studying the human brain.

To overcome such obstacles, scientists created a very interesting combined model: Human-mouse chimera¹⁹. Researchers introduce human neural progenitor cells (NPCs) into mouse embryos. These cells are derived from patients with microcephaly, allowing scientists to observe how the condition develops in a living organism²⁰. The human NPCs integrate into the mouse brain, developing alongside the mouse’s neural cells. This chimeric model enables the study of human-specific aspects of brain development and the differences arising from microcephaly mutations²¹. By comparing the development of these human NPCs with those derived from healthy individuals, researchers can identify specific developmental abnormalities associated with microcephaly, such as premature differentiation or altered cell proliferation  The chimeric model allows for detailed genetic and molecular analysis of the affected human cells within the mouse brain environment²². Researchers can study the expression of key genes and proteins involved in brain development and identify potential pathways disrupted in microcephaly. This model provides a platform for testing potential therapeutic interventions. Researchers can introduce drugs or gene therapies to the chimeric embryos or postnatal mice to see if they can correct or mitigate the defects caused by microcephaly-associated mutations²³. Overall, human-mouse chimeras offer a powerful tool for studying microcephaly in a more complex and physiologically relevant context than in vitro models, providing insights that can lead to the development of effective treatments.

The primary limitation of the animal model is its inability to study features specific to humans, as these features cannot be replicated in animal models²⁴. For example, one of the most obvious differences between humans and mice is the abundance of basal radial glial cells (bRG) in human fetal tissue, which account for most of the generation of neurons in the upper nuclear layer²⁵. In addition, rare bRG cells present in mice may not possess the same proliferative capacity observed in humans²⁶. Secondly, animal models are unable to model polygenic diseases, or diseases with or without known causative mutations, which are difficult to model in mice²⁷. Unlike animal models, brain organoids have the ability to be manipulated physically and chemically, allowing the effects of various molecules on their development to be tested in a controlled environment²⁸. For example, the reproducibility of the organoids is more easy to control than the animal models. The “environment” of organoids is easy to control. While animal models are more complex systems their well-being is not only influenced by the environment that kept the animal, but also their psychological state, food, physical health and etc. Moreover, a large number of animal experiments may result in an ethical problem. Organoids can avoid these ethical limitations and grow in large numbers, providing a higher efficiency in research.

1.2 Cerebral Organoids

1.2.1 Pluripotent Stem Cells (PSCs)

Pluripotent stem cells can partially reproduce a teratoma that contains a variety of semi-organized tissues following uncontrolled differentiation and self-organization. The teratoma is a tumor-like formation containing tissue belonging to all 3 germ layers, and it is formed when the differentiation process of a pluripotent stem cell has been disrupted²⁹. Pluripotent stem cells can partially reproduce a teratoma that contains a variety of semi-organized tissues following uncontrolled differentiation and self-organization, the enriched PCSs through the process of forming a teratoma can be separated to generate specific organoids³⁰. When injected into an immunocompromised animal, these cells can form teratomas, which are tumors that contain a variety of differentiated cell types from all three germ layers. This is because pluripotent stem cells can differentiate into any cell type, and in the absence of proper regulatory signals, they can form disorganized structures³¹. Teratomas validate the pluripotency of stem cells, as they encompass various tissue types derived from all three germ layers. This feature is essential for creating organoids, such as cerebral organoids, used to model brain development and disease.

The production of cerebral organoids showcases the controlled differentiation potential of pluripotent stem cells, which could form teratomas if not regulated³². By providing proper 3D scaffold and biochemical factors which include basic fibroblast factors, epidermal growth factors, retinoic acid, and inhibitors of SMAD signaling to induce and maintain neural progenitor populations, Lancaster et al. differentiated cells from PSCs can self-organize to tissue-specific organoids which includes brain organoids³³. When stem cells are placed within a hydrogel, often in a 3D matrix called Matrigel, they can assemble into structures and also develop into organized clusters. Exogenous factors, such as growth factors, signaling molecules, scaffolds, and mechanical stimulation, play a crucial role in directing the differentiation and organization of stem cells into organoids. For instance, basic fibroblast growth factor (bFGF) and retinoic acid (RA) help guide neural progenitors, while bone morphogenetic protein (BMP) inhibitors promote neural induction by preventing mesodermal differentiation³⁴.

Pluripotent stem cells have the potential to differentiate into a wide range of tissues and organoids derived from the three primary germ layers: ectoderm–neural cells, mesoderm–cardiomyocytes and hematopoietic cells, and endoderm–pancreatic cells³⁵. The usage of PSCs-grown liver organoids and lung organoids has helped a lot in the research of different kinds of diseases³⁶.

In the study of microcephaly, cerebral organoids, which are grown from induced pluripotent stem cells, are frequently³⁷. With the intrinsic Properties of PSCs discussed above, such as developmental potential, the PSCs can differentiate into neurons and glial cells, which are essential for forming brain tissue. Moreover, with the ability to form cell-cell interactions, these cells can facilitate the formation of complex structures similar to those in the developing brain^32,11. Embedding stem cells in a 3D matrix, such as Matrigel, provides a scaffold that supports cell attachment and growth, enabling the formation of three-dimensional structures, also mimicking the in vivo environment, and promoting more realistic tissue development³⁸. Other aspects promote the overall ability of cerebral organoids generated from PSCs. Specific growth factors and signaling molecules guide the differentiation of stem cells into various neural cell types and promote the regional specification seen in the brain³⁹. Sequential addition of temporal regulation over time mimics the developmental timeline leading to the formation of different brain regions⁴⁰. The formation of gradients of morphogens (e.g., RA, BMP inhibitors) within the organoid can lead to spatial organization similar to in vivo brain development. Cells within the organoid undergo processes like cell sorting and regionalization, leading to the spontaneous formation of layered structures and distinct brain regions (e.g., cerebral cortex, ventricles).

Progress in recent research of in vitro models of organ systems has marked an enormous capacity of self-organizing in the development from pluripotent stem cells to whole tissues¹¹. Derived from human pluripotent stem cells, cerebral organoids are three-dimensional in vitro models that can be utilized to investigate the complex and human-specific features of early brain development¹¹. COs, also known as cerebral organoids, can be used to dissect the underlying mechanisms of microcephaly while also helping to elucidate the fundamental mechanism of normal human brain development^41,42.  Microcephaly is thought to arise from a common disease mechanism ultimately owing to dysregulation of the cell cycle that disrupts the timing of this carefully orchestrated neurogenesis⁴³. Organoids generated from pluripotent stem cells derived from microcephaly patients with mutations in mitosis-associated genes show dysregulation of the cell division plane, resulting in premature depletion of NPCs (neural progenitor cells) and the formation of smaller organoids^44,45. A recent study modeling a pathogenic AUTS2 missense variant in COs revealed significantly reduced organoid growth⁴⁶. AUTS2 is one of the genes that can cause microcephaly once mutant. AUTS2 mutant COs showed dysregulated cell cycle control and reduced symmetrical (horizontal) cellular division, which correlated with premature neuronal differentiation in comparison to control COs. Increased asymmetrical progenitor divisions and premature neuronal differentiation are also common features observed in other CO models containing mutant microcephaly genes⁴⁷.

During the research of cerebral organoids, several essential technologies were involved in developing the organoid. Single-cell RNA-seq, combined with immunohistochemical, spatial transcriptomic, and chromatin immunoprecipitation techniques can provide important morphological context to reconstruct the organization of disease-related expression patterns⁴⁸. Single-cell RNA sequencing enables researchers to identify and characterize the array of cell types found within brain organoids. This is vital in understanding the organoid’s cellular composition and the dynamic changes throughout its development. By assessing gene expression at the individual cell level, single-cell RNA-seq can determine the lineage relationships between different cell types⁴⁹. This reveals how pluripotent stem cells differentiate into various neural and glial cell types. Single-cell RNA-seq offers insights into the activation of distinct developmental pathways and gene networks during various stages of organoid development. This aids in comprehending the regulation of these pathways and their role in forming brain-like structures. Researchers can monitor changes in gene expression over time, which enables them to examine the progression of organoid development and pinpoint crucial moments for significant developmental events⁵⁰. Moreover, single-cell RNA sequencing (scRNA-seq) can detect unusual gene expression patterns and disrupted pathways in organoids, which are derived from patient-specific induced pluripotent stem cells (iPSCs). This aids in understanding the molecular mechanisms that underpin the disease. By comparing scRNA-seq data from healthy and diseased organoids, researchers can identify specific genes and pathways with differential expression. This provides potential targets for therapeutic intervention⁵¹.

Early rosette structures in COs shape ventricular zone-like structures that are comprised of stratified progenitors, which undergo distinct stromal translocations critical to their progression through the cell cycle. Apical neural progenitors divide symmetrically within these structures in COs, but during neurogenesis, they shift to asymmetric divisions, forming another apical neural progenitor and either a neuron or an intermediate progenitor (IP) cell, which is a type of basal neural progenitor⁵². In contrast to rodents, humans and larger mammals possess a significant population of outer radial glial cells in the outer subventricular zone (OSVZ), which serve as a plentiful source of IP cells¹³,⁵³.

2. Modelling microcephaly using organoids

2.1 Usage of Organoids in the Research of Primary Microcephaly

In the pioneering study that involved the usage of cerebral organoids in microcephaly, Lancaster and Knoblish reprogrammed patients’ cells that carried mutations in CKD5RAP2, a protein whose mutation often causes microcephaly, into induced pluripotent cells and then generated organoids⁵⁴. After that, Biel et al observed that the mutant cerebral organoids displayed smaller neuroepithelial regions and contained fewer progenitor cells and more neurons, suggesting that a premature differentiation had taken place. The authors also found more oblique divisions of the aRG cells, which would lead to their delamination. While this phenomenon is a natural event in the human brain that generates bRG cells, its early occurrence would lead to an impaired tangential expansion of the neuroepithelium⁵⁵.

Mutations in the ASPM gene (abnormal spindle-like microcephaly-associated) are the most common cause of primary microcephaly in humans⁵⁶. In the research of Li et al, ASPM patient-derived cerebral organoids were also shown to display severe growth defects. However, limitations of available transgenic ferret lines have brought up a big problem. The organoids were extremely disorganized with barely recognizable neuroepithelial regions in which proliferation rates were very low.          

Mutations in the gene coding for centrosomal-associated-P4.1 protein (CPAP) cause Seckel syndrome: primary microcephaly that is coupled with a reduction in body size. Using a combination of patient fibroblasts, iPS-derived neural stem cells (NSCs), and cerebral organoids, Gabriel and colleagues identified a role for CPAP in primary cilia disassembly and control of the progenitor cell pool⁵⁷. Mutated CPAP did not localize to the cilia, therefore impairing its disassembly. In agreement with the ciliary defects, the authors furthermore identified defective cell cycle progression and premature differentiation. Based on these observations, they proposed that, upon CPAP mutation, impaired cilia disassembly leads to the reduced proliferation of progenitor cells and, as a consequence, to their premature cell cycle exit and differentiation⁵⁸,⁵⁹.

An entirely different molecular cause of microcephaly was recently highlighted through the study of the microcephaly-associated factor NARS1, a Class IIa tRNA synthetase. These patients displayed severe microcephaly, cerebellar atrophy, and ventriculomegaly. Mutations affected NARS1 stability, leading to global protein synthesis defects. The growth of these organoids was severely affected, with impaired formation of the neuroepithelial structures (rosettes). NARS1 mutations led to the reduced proliferation of neuronal progenitors, leading to reduced neuronal production. Moreover, increased levels of apoptosis were also reported. Surprisingly, single-cell RNA-seq data pointed to an altered cell fate towards astrocytes at the expense of neurons in mutant organoids⁶⁰.

2.2 Role of Organoid in the Research of Secondary Microcephaly

Secondary microcephaly, which by definition is postnatal, results from the alteration of later and more diverse mechanisms than primary microcephaly, related to brain maturation after birth. Usually, syndromic, secondary microcephaly is often only one of many clinical signs in multi-organ pathologies¹⁷.

Developmental and epileptic encephalopathies (DEE), a group of disorders that are characterized by intractable epileptiform activity and impaired cerebral functions, are sometimes associated with secondary microcephaly⁶¹. Using patient-derived iPSCs that were mutated in WWOX (an abbreviation of the name of an enzyme and associated gene called “WW domain-containing oxidoreductase” that was associated with a severe infantile epileptic encephalopathy), a gene encoding the WW domain-containing oxidoreductase that was associated with a severe infantile epileptic encephalopathy, Steinberg et al. generated cerebral organoids aged between 10 and 24 weeks and identified impaired expression of the cortical markers TBR1, CTIP2, SATB2, and layering defects that progressively worsened at week 24⁶². These defects were correlated with an activation of Wnt signaling, an ancient and evolutionarily conserved pathway that regulates crucial aspects of cell fate determination, cell migration, cell polarity, neural patterning and organogenesis during embryonic development. WWOX-depleted organoids further exhibited hyperexcitability, likely due to a disrupted balance between glutamatergic and GABAergic neurons, the receptors of Gmma-aminobutyric acid in the central nervous system⁶³.

Cohen syndrome is a rare autosomal recessive golgipathy that is characterized by motor delays, retinal dystrophy appearing by mid-childhood, progressive severe myopia, hypotonia, joint hypermobility, and secondary microcephaly that is associated with intellectual disability⁶⁴,⁶⁵.

It results from loss of function mutations in the VPS13B gene, which regulates vesicle-mediated protein sorting and transport. Mutations in the VPS13B gene, which encodes the VPS13B protein (also known as COH1), are responsible for Cohen syndrome. The VPS13B protein plays a crucial role in various cellular processes, and its dysfunction due to mutations can disrupt these processes, leading to the clinical manifestations of Cohen syndrome. More recently, recycling and internalization assays in HeLa cells have shown another role of VPS13B in transport from early endosomes to recycling endosomes via interaction with Stx6- and Stx13-containing vesicles⁶⁶. VPS13B mutations affect the proper development and function of neutrophils, contributing to chronic neutropenia observed in Cohen syndrome. This immunodeficiency results from defective vesicular trafficking within the hematopoietic system, affecting the survival and maturation of neutrophils⁶⁷. Moreover, when VPS13B is expressed in neurons, it contributes to synaptic vesicle recycling. Disruptions in this process affect neurotransmitter release and synaptic signaling, which may explain the intellectual disability and other neurological symptoms of Cohen syndrome⁶⁸.  Patient-derived iPSCs were differentiated into either forebrain-like glutamatergic excitatory neurons in 2D cultures or 3D neurospheres using dual SMAD inhibition⁶⁹.

Discussion

Limitation of Organoids

Although hereditary microcephaly syndrome is relatively rare, examining these disorders offers unique advantages because they can reveal in unprecedented detail the molecular mechanisms that determine NPCS (Neural progenitor cells) maintenance, brain development, and human brain evolution⁷⁰.  However, these diseases need to be studied in a system that closely matches the human brain which is still a challenge. In this context, recent advances in brain organoids have laid a strategic position in the field of microcephaly research.  Guhr and his team identified gaps in current cortical organoid models, noting the underrepresentation of crucial brain cells like interneurons, microglia, and endothelial cells. Interneurons, which migrate from specific brain regions to the cortex, are critical for cortical development, but their migration is not well-modeled in organoids. Additionally, the absence of microglia, brain-resident immune cells, and endothelial cells, which form the vascular network, limits the model’s ability to replicate the in vivo brain environment, particularly in studies of neuroinflammation and neurovascular interactions⁷¹.

One of the major challenges of the organoid model for the study of brain development is that it may faithfully reproduce human neurogenesis at early stages of neurogenesis, but much less at late stages, such as the lack of certain cell types, for example, microglia and interneuron, makes it challenging to model postnatal microcephaly mechanisms. These later stages of development, crucial for understanding brain migration and synaptogenesis, are under-represented in most current organoid protocols, limiting the utility of the model in postnatal development research⁷². In contrast, the more advanced stages of development that are valuable in understanding the mechanisms of migration, synaptogenesis, and brain maturation, largely associated with postnatal microcephaly, are much more difficult to model. In particular, this is because certain derivatives are either somewhat or not represented in most of the current cortical organoid protocols⁷¹. Likewise, populations of interneurons, which appear in specific brain areas and then migrate to other regions such as the cortex, are under-represented in cortical organoid models. Microglial cells, which are the brain-resident macrophages acting as the immune system of the brain, or endothelial cells and pericytes, which make up the vascular network that carries and provides nutrients and oxygen throughout the whole brain, have a mesodermal origin and are also not present in current cortical models.

Another issue of organoids might be the heterogeneity of the three-dimensional tissue that is generated from hiPSCs could compromise the reproducibility of differentiation experiments, including human cortical organoid generation⁷³,⁷⁴. In the study of Ichismima and his team, they highlighted that some hiPSC lines are predisposed to certain differentiation pathways due to their epigenetic memory, which affects reproducibility in experiments. This variability complicates the use of hiPSCs for studying neurodevelopmental disorders like microcephaly, as different cell lines may yield different outcomes under the same experimental conditions, affecting the fidelity of the organoid model⁷⁵. This could explain why some cell lines are more prone to undergo neural differentiation than others. This could also explain the variable developmental outcomes in the type of neural identities that are acquired among cell lines within a given protocol.

Future Development of the Model

Comprehensive decoding of the mechanisms of microcephaly requires a repertoire of mutant models, which is the significant bottleneck at the current state of the art. Thus generating a repertoire of iPSCs from microcephaly patients will enable us to generate patient-specific 3D tissues. Organoid generation is less critical than acquiring stable iPSCs that harbor microcephaly mutations⁷⁶.

An attractive alternative to patient-specific iPSCs is the genome tailoring to acquire disease-relevant patient mutations in pluripotent cells. Of note, CRISPR–Cas9-based genome editing has not been sufficiently utilized in microcephaly research using brain organoids except for a recent report, where the authors have successfully eliminated the tight junction protein in human embryonic stem cells⁷⁷. In this regard, CRISPR/Cas9-edited organoids displayed early neuronal differentiation and reduced progenitors.

Remarkably, comparative studies employing both mouse and human NPCs uncovered that human NPCs were more severely affected. Thus, applying genome tailoring in hydrogels to obtain organoids with patient-specific mutations will serve as a powerful tool and will allow us to generate microcephaly brain organoids to conduct a functional analysis of candidate genes in healthy human brain development.

Methodology

A comprehensive literature search was conducted to gather relevant studies on the use of brain organoids in researching genetically induced microcephaly. The databases used included PubMed, Web of Science, and Google Scholar to identify peer-reviewed articles, reviews, and research papers published up to July 2024. Keywords for the search were “brain organoids,” “microcephaly,” “genetically induced microcephaly,” “pluripotent stem cells,” “cerebral organoids,” and “neurogenesis.” The search was refined by including specific genes and pathways involved in microcephaly, such as MCPH1 and AUTS2.

Studies were included if they met the following criteria: focusing on the development or application of brain organoids in microcephaly research; investigating genetically induced microcephaly using patient-derived organoids or animal models; being published in English and peer-reviewed. Exclusion criteria were studies not directly addressing the use of organoids in microcephaly research, conference abstracts, editorials, opinion pieces, and those lacking sufficient methodological detail.

Conclusion

Brain organoid technology, particularly cortical organoid models, has come a long way in less than 10 years and shows its rich potential in simulating microcephaly and providing valuable information for understanding the mechanisms of human brain development. The next few years are likely to see further substantial improvements that could homogenize differentiation and allow routine studies of advanced stages of brain maturation. In this regard, the field of microfluidics has begun to interact with organoid technology with the aim of using biomechanical Microsystems to control the fluid environment of organoids, thereby increasing their maturity. Considering environmental parameters is even more important because complex interactions between genetic factors and environmental constraints may partly explain the clinical heterogeneity observed among people with microcephaly, sometimes with mutations in the same gene, and the significant differences in their cognitive outcomes. There is no doubt that models with higher quality, longevity and reproducibility, as well as a microenvironment that closely mimics brain-specific extracellular matrix cues and allows for control of exogenous factors, will improve our understanding of the mechanisms of microcephaly and provide new strategies for screening molecules to improve cognitive outcomes in these patients.

Acknowledgments

Thank you for the guidance of  Klara Aliz Stark, mentor from Cambridge University in the development of this research paper.

Reference

1. Xiaolei Yin, Benjamin E Mead , Helia Safaee, Robert Langer, Jeffrey M Karp, Oren Levy，Engineering Stem Cell Organoids.Cell Stem Cell, 25-38(2016). [↩]
2. Hansen, D. V., Lui, J. H., Parker, P. R. L., & Kriegstein, A. R,Neurogenic radial glia in the outer subventricular zone of human neocortex. Nature, 554-561(2010). [↩]
3. Lewitus, E., Kelava, I., & Huttner, W. B, Conical expansion of the outer subventricular zone and the primate brain evolution.Current Opinion in Neurobiology,1208-1214(2013). [↩]
4. Cunningham, C. L., Martínez-Cerdeño, V., & Noctor, S. C, Microglia regulate the number of neural precursor cells in the developing cerebral cortex. The Journal of Neuroscience, 4216-4233(2013). [↩]
5. Lancaster, M.A., Knoblich, J.A, Generation of cerebral organoids from human pluripotent stem cells. Nat. Protoc, 2329–2340(2014). [↩] [↩] [↩]
6. Ichisima, J., Suzuki, N.M.; Samata, B., Awaya, T.; Takahashi, J., Hagiwara, M., Nakahata, T., Saito, M.K, Verification and rectification of cell type-specific splicing of a Seckel syndrome-associated ATR mutation using iPS cell model. J. Hum. Genet,  445–458(2019). [↩]
7. Xiaolei Yin, Benjamin E Mead , Helia Safaee, Robert Langer, Jeffrey M Karp, Oren Levy,Engineering Stem Cell Organoids.Cell Stem Cell, 25-38(2016). [↩] [↩]
8. Eiraku M, Takata N, Ishibashi H, Kawada M, Sakakura E, Okuda S, Sekiguchi K, Adachi T, Sasai Y, Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature, 51–56(2011). [↩]
9. Li L, Xie T, Stem cell niche: structure and function. Annu Rev Cell Dev Biol, 605–631(2005). [↩]
10. Lancaster M.A, Renner M; Martin C.A, Wenzel D, Bicknell L.S, Hurles M.E, Homfray T, Penninger J.M, Jackson A.P, Knoblich J.A, Cerebral organoids model human brain development and microcephaly. Nature, 373–379(2013). [↩]
11. Lancaster M.A, Renner M; Martin C.A, Wenzel D, Bicknell L.S, Hurles M.E, Homfray T, Penninger J.M, Jackson A.P, Knoblich J.A, Cerebral organoids model human brain development and microcephaly. Nature,  373–379(2013). [↩] [↩] [↩] [↩]
12. Takasato M, Er PX, Becroft M, Vanslambrouck JM, Stanley EG, Elefanty AG, Little MH, Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney. Nat Cell Biol, 118-26(2014). [↩]
13. Lancaster M.A, Renner M; Martin C.A, Wenzel D, Bicknell L.S, Hurles M.E, Homfray T, Penninger J.M, Jackson A.P, Knoblich J.A, Cerebral organoids model human brain development and microcephaly. Nature, 373–379(2013). [↩] [↩]
14. Gabriel E, Ramani A., Karow U, Gottardo M, Natarajan K, Gooi L M, et al,  Recent Zika Virus isolates induce premature differentiation of neural progenitors in human brain organoids. Cell Stem Cell, 397–406(2017). [↩]
15. Li J, Yuan Y, Liu C, Xu Y, Xiao N, Long H, Luo Z, Meng S, Wang H, Xiao B, Mao X, Long L, DNAH14 variants are associated with neurodevelopmental disorders. Hum Mutat, 940-949(2022). [↩]
16. Biel A, Castanza AS, Rutherford R, Fair SR, Chifamba L, Wester JC, Hester ME and Hevner RF, AUTS2 Syndrome: Molecular Mechanisms and Model Systems. Front. Mol. Neurosci,15:858582(2022). [↩]
17. Gabriel E, Ramani A, Altinisik N, Gopalakrishnan J, Human Brain Organoids to Decode Mechanisms of Microcephaly. Front Cell Neurosci, 14:115(2020). [↩] [↩]
18. Farcy, S.; Albert, A.; Gressens, P.; Baffet, A.D.; El Ghouzzi, V. Cortical Organoids to Model Microcephaly. Cells. 2022, 11, 2135. https://doi.org/10.3390/ cells11142135 [↩]
19. Marthaler, A. G., & Fernández, M. F, Human-Mouse Chimeric Models of Neurodevelopmental Disorders. Trends in Neurosciences, 563-577(2021). [↩]
20. Schmitt K, Curlin JZ, Remling-Mulder L, Aboellail T, Akkina R, Zika virus-induced microcephaly and aberrant hematopoietic cell differentiation modeled in novel neonatal humanized mice. Front Immunol,14:1060959(2013). [↩]
21. Schmitt K, Curlin JZ, Remling-Mulder L, Aboellail T, Akkina R, Zika virus-induced microcephaly and aberrant hematopoietic cell differentiation modeled in novel neonatal humanized mice. Front Immunol, 14:1060959( 2023). [↩]
22. Marthaler, A. G., & Fernández, M. F, Human-Mouse Chimeric Models of Neurodevelopmental Disorders. Trends in Neurosciences, 563-577(2021 [↩]
23. Schmitt K, Curlin JZ, Remling-Mulder L, Aboellail T, Akkina R, Zika virus-induced microcephaly and aberrant hematopoietic cell differentiation modeled in novel neonatal humanized mice. Front Immunol, 14:1060959(2023). [↩]
24. Biel A, Castanza AS, Rutherford R, Fair SR, Chifamba L, Wester JC, Hester ME and Hevner RF, AUTS2 Syndrome: Molecular Mechanisms and Model Systems. Front. Mol. Neurosci, 15:858582(2022). [↩]
25. Nowakowski TJ, Salama SR, Cerebral Organoids as an Experimental Platform for Human Neurogenomics. Cells,11(18):2803(2022). [↩]
26. Wang, H, Modeling Neurological Diseases With Human Brain Organoids. Front Synaptic Neurosci, 8,10, 15(2018). [↩]
27. Biel A, Castanza AS, Rutherford R, Fair SR, Chifamba L, Wester JC, Hester ME and Hevner RF, AUTS2 Syndrome: Molecular Mechanisms and Model Systems. Front. Mol Neurosci, 15:858582(2022). [↩]
28. Xiaolei Yin, Benjamin E Mead , Helia Safaee, Robert Langer, Jeffrey M Karp, Oren Levy, Engineering Stem Cell Organoids. Cell Stem Cell, 25-38(2016). [↩]
29. Tatyana A Prokhorova, Linda M Harkness, Ulrik Frandsen, Nicholas Ditzel, Henrik D Schrøder, Jorge S Burns, Moustapha Kassem, Teratoma formation by human embryonic stem cells is site dependent and enhanced by the presence of Matrigel.Stem Cells Dev, 47-54(2009). [↩]
30. Przyborski SA, Differentiation of human embryonic stem cells after transplantation in immune-deficient mice.Stem Cells,  1242-50(2005). [↩]
31. Yamanaka, S,  Strategies and new developments in the generation of patient-specific pluripotent stem cells. Cell Stem Cell, 39-49(2007). [↩]
32. Lancaster, M.A., Knoblich, J.A, Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc, 2329–2340(2014). [↩] [↩]
33. Lancaster, M.A., Knoblich, J.A, Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc,  2329–2340(2014). [↩]
34. Lancaster, M.A., Knoblich, J.A, Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc,  2329–2340(2014 [↩]
35. Takahashi K,  Yamanaka S, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 663-676(2006). [↩]
36. Xiaolei Yin, Benjamin E Mead , Helia Safaee, Robert Langer, Jeffrey M Karp, Oren Levy, Engineering Stem Cell Organoids.Cell Stem Cell,  25-38( 2016). [↩]
37. Farcy, S, Albert A, Gressens P, Baffet AD, El Ghouzzi V, Cortical Organoids to Model Microcephaly. Cells,  2135(2022). [↩]
38. Lancaster M.A, Renner M, Martin CA, Wenzel D, Bicknell L.S, Hurles M.E, Homfray T, Penninger J.M, Jackson A.P, Knoblich J.A, Cerebral organoids model human brain development and microcephaly. Nature,  373–379(2013). [↩]
39. Qian X, Jacob F, Song M.M, Nguyen H.N, Song H, Ming G L, Generation of human brain region-specific organoids using a miniaturized spinning bioreactor. Nat Protoc, 565–580(2018. [↩]
40. Qian X, Jacob F, Song M.M, Nguyen H.N, Song H, Ming G L, Generation of human brain region-specific organoids using a miniaturized spinning bioreactor. Nat Protoc, 565–580(2018). [↩]
41. Gabriel E, Ramani A, Altinisik N, Gopalakrishnan J, Human Brain Organoids to Decode Mechanisms of Microcephaly. Front Cell Neurosci,  8;14:115(2020). [↩]
42. Gopalakrishnan J,  The emergence of stem cell-based brain organoids: trends and challenges. Bioessays,  41:e1900011(2019). [↩]
43. Jean F, Stuart A, and Tarailo-Graovac M, Dissecting the genetic and etiological causes of primary microcephaly. Front.Neurol, 11:570830(2020). [↩]
44. Lancaster M.A, Renner M; Martin C.A, Wenzel D, Bicknell L.S, Hurles M.E, Homfray T, Penninger J.M, Jackson A.P, Knoblich J.A, Cerebral organoids model human brain development and microcephaly. Nature, 373–379( 2013). [↩]
45. Gabriel E, Ramani A., Karow U, Gottardo M, Natarajan K, Gooi L M, et al，  Recent Zika Virus isolates induce premature differentiation of neural progenitors in human brain organoids. Cell Stem Cell，397–406(2017). [↩]
46. Fair SR, Schwind W, Julian D, Biel A, Ramadesikan S, Westfall J, etal, Cerebral organoids containing an AUTS2 missense variant model microcephaly. Brain, 387-404(2023). [↩]
47. Biel A, Castanza AS, Rutherford R, Fair SR, Chifamba L, Wester JC, Hester ME and Hevner RF, AUTS2 Syndrome: Molecular Mechanisms and Model Systems. Front Mol Neurosci, 15:858582( 2022). [↩]
48. Camp JG, Badsha F, Florio M, Kanton S, Gerber T, Wilsch-Bräuninger M, Lewitus E, Sykes A, Hevers W, Lancaster M, Knoblich J A, Lachmann R, Pääbo S, Huttner W B, & Treutlein B,  Human cerebral organoids recapitulate gene expression programs of fetal neocortex development. Proceedings of the National Academy of Sciences,15672-15677(2015). [↩]
49. Gabriel E, Ramani A, Altinisik N, Gopalakrishnan J, Human Brain Organoids to Decode Mechanisms of Microcephaly. Front Cell Neurosci, 14:115(2020). [↩]
50. Wang X, Tsai JW, LaMonica B, Kriegstein AR,  A new subtype of progenitor cell in the mouse embryonic neocortex. Nat. Neurosci, 555–561(2011). [↩]
51. Agnieska Brazovskaja, Barbara Treutlein, J Gray Camp, High-throughput single-cell transcriptomics on organoids.Curr Opin Biotechnol, 167-171(2019). [↩]
52. Pebworth MP, Ross J,Andrews M, Bhaduri A,and Kriegstein AR, Human intermediate progenitor diversity during cortical development. Pro. Natl. Acad. Sci, e2019415118(2021). [↩]
53. Kadoshima T, Sakaguchi H, Nakano T, Soen M, Ando S, Eiraku M, Sasai Y, Self-organization of axial polarity, inside-out layer pattern, and species-specific progenitor dynamics in human ES cell-derived neocortex. Proc Natl Acad Sci, 20284–20289(2013). [↩]
54. Lancaster MA, Knoblich JA, Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc, 2329–2340(2014). [↩]
55. Biel A, Castanza AS, Rutherford R, Fair SR, Chifamba L, Wester JC, Hester ME and Hevner RF, AUTS2 Syndrome: Molecular Mechanisms and Model Systems. Front. Mol Neurosci,   15:858582(2022). [↩]
56. Letard P, Drunat S, Vial Y, Duerinckx S, Ernault A, Amram D, Arpin S, Bertoli M, Busa T, Ceulemans B, et al, Autosomal recessive primary microcephaly due to ASPM mutations: An update. Hum Mutat,  319–332(2018). [↩]
57. Gabriel E, Wason A, Ramani A, Gooi LM, Keller P, Pozniakovsky A, Poser I, Noack F, Telugu NS, Calegari F, et al, CPAP promotes timely cilium disassembly to maintain neural progenitor pool. EMBO J,  803–819(2016). [↩]
58. Lange C, Huttner WB, Calegari F, Cdk4/cyclinD1 overexpression in neural stem cells shortens G1, delays neurogenesis, and promotes the generation and expansion of basal progenitors. Cell Stem Cell, 320–331(2009). [↩]
59. Pilaz LJ, Patti D, Marcy G, Ollier E, Pfister S, Douglas RJ, Betizeau M, Gautier E, Cortay V, Doerflinger N, et al, Forced G1-phase reduction alters mode of division, neuron number, and laminar phenotype in the cerebral cortex. Proc Natl Acad Sci,  21924–21929( 2009). [↩]
60. Wang L, Li Z, Sievert D, Smith DEC, Mendes MI, Chen DY, Stanley V, Ghosh S, Wang Y, Kara M, et al, Loss of NARS1 impairs progenitor proliferation in cortical brain organoids and leads to microcephaly. Nat Commun,  4038(2020). [↩]
61. McTague A, Howell KB, Cross JH, Kurian MA, Scheffer IE, The genetic landscape of the epileptic encephalopathies of infancy and childhood.Lancet Neurol, 304-16(2016). [↩]
62. Steinberg DJ, Repudi S, Saleem A, Kustanovich I, Viukov S, Abudiab B, Banne E, Mahajnah M, Hanna JH, Stern S, et al, Modeling genetic epileptic encephalopathies using brain organoids. EMBO Mol Med.e13610( 2021). [↩]
63. Steinberg DJ, Repudi S, Saleem A, Kustanovich I, Viukov S, Abudiab B, Banne E, Mahajnah M, Hanna JH, Stern S, et al, Modeling genetic epileptic encephalopathies using brain organoids. EMBO Mol Med, e13610(2021). [↩]
64. Passemard S, Perez F, Colin-Lemesre E, Rasika S, Gressens P, El Ghouzzi V, Golgi trafficking defects in postnatal microcephaly: The evidence for “Golgipathies”. Prog Neurobiol, 46–63( 2017). [↩]
65. Chandler KE, Kidd A, Al-Gazali L, et al, Diagnostic criteria, clinical characteristics, and natural history of Cohen syndrome. Journal of Medical Genetics,233-241( 2003). [↩]
66. Koike S, Jahn R, SNAREs define targeting specificity of trafficking vesicles by combinatorial interaction with tethering factors.Nat Commun, 1608(2019). [↩]
67. Kolehmainen J, Black GCM, Saarinen A, et al, Cohen syndrome is caused by mutations in the novel gene COH1, encoding a transmembrane protein with a presumed role in vesicle-mediated sorting and intracellular protein transport. American Journal of Human Genetic, 123-130(2004). [↩]
68. Velayos-Baeza A, Vettori A, Copley RR, & Monaco AP, Analysis of the human VPS13 gene family. Genomics, 536-549(2004). [↩]
69. Lee YK, Hwang SK, Lee SK, Yang J, Kwak JH, Seo H, Ahn H, Lee YS, Kim J, Lim CS, et al, Cohen Syndrome Patient iPSC-Derived Neurospheres and Forebrain-Like Glutamatergic Neurons Reveal Reduced Proliferation of Neural Progenitor Cells and Altered Expression of Synapse Genes. J Clin Med, 1886(2020). [↩]
70. Shi Y,Sun L, Wang M, Liu J, Zhong S, Li R, Li P, Guo L, Fang A, Chen R, et al, Vascularized human cortical organoid model cortical development in vivo. PLoS Biol,  e3000705(2020). [↩]
71. Anke Guhr, Sabine Kobold, Stefanie Seltmann, Andrea E M Seiler Wulczyn, Andreas Kurtz , Peter Löser, Recent Trends in Research with Human Pluripotent Stem Cells: Impact of Research and Use of Cell Lines in Experimental Research and Clinical Trials. Stem Cell Rep, 485–496(2018). [↩] [↩]
72. Chahrour M, Zoghbi HY, The Story of Rett Syndrome: From Clinic to Neurobiology. Neuron, 422–437(2007). [↩]
73. Lancaster MA, Renner M, Martin CA, Wenzel D, Bicknell LS, Hurles ME, Homfray T, Penninger JM, Jackson AP, Knoblich JA, Cerebral organoids model human brain development and microcephaly. Nature, 373–379( 2013). [↩]
74. Qian X, Nguyen HN, Song MM, Hadiono C, Ogden SC, Hammack C, Yao B, Hamersky GR, Jacob F, Zhong C, et al, Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure. Cell, 1238–1254(2016). [↩]
75. Ichisima J, Suzuki NM, Samata B, Awaya T, Takahashi J, Hagiwara M, Nakahata T, Saito MK, Verification and rectification of cell type-specific splicing of a Seckel syndrome-associated ATR mutation using iPS cell model. J Hum Genet,  445–458(2019). [↩]
76. Gopalakrishnan J,  The emergence of stem cell-based brain organoids: trends and challenges. Bioessays,e1900011(2019). [↩]
77. Bendriem RM, Singh S, Aleem AA, Antonetti DA, Ross ME. Tight junction protein occludin regulates progenitor Self-Renewal and survival in developing cortex.eLife, 49376(2019). [↩]