Shedding light on latent pathogenesis and pathophysiology of mental disorders: The potential of iPS cell technology

Abstract

Mental disorders are considered as one of the major healthcare issues worldwide owing to their significant impact on the quality of life of patients, causing serious social burdens. However, it is hard to examine the living brain—a source of psychiatric symptoms—at the cellular, subcellular, and molecular levels, which poses difficulty in determining the pathogenesis and pathophysiology of mental disorders. Recently, induced pluripotent stem cell (iPSC) technology has been used as a novel tool for research on mental disorders. We believe that the iPSC‐based studies will address the limitations of other research approaches, such as human genome, postmortem brain study, brain imaging, and animal model analysis. Notably, studies using integrated iPSC technology with genetic information have provided significant novel findings to date. This review aimed to discuss the history, current trends, potential, and future of iPSC technology in the field of mental disorders. Although iPSC technology has several limitations, this technology can be used in combination with the other approaches to facilitate studies on mental disorders.

Mental disorders are recognized as one of the major healthcare problems worldwide because of their significant impact on the quality of life of patients, resulting in significant social burdens. However, it is difficult to examine the living brain—a source of psychiatric symptoms—at the cellular, subcellular, and molecular levels. Therefore, in general, various approaches, such as human genome, postmortem brain study, brain imaging, and animal model analysis, have been used to investigate the pathogenesis and pathophysiology of mental disorders. Each of these models has its advantages and disadvantages (see Fig. 1).

Fig. 1.

Comparison of the existing approaches for the research on mental disorders.

The advantage of the genetic study is its ability to determine the full diversity of mental disorders in an unbiased manner. Notably, large‐scale human genetic studies have identified many genetic variants that are significantly associated with the onset of mental disorders. Moreover, mental disorders, such as schizophrenia (SCZ), autism spectrum disorders (ASD), and bipolar disorder, are known to share risk genetic variants regardless of their clinical manifestations. ¹ , ² These findings indicate genetic heterogeneity and complexity in mental disorders. However, genetic studies cannot validate the biological and functional significance of the identified genetic variants in the brain; moreover, the dynamic pathogenesis and pathophysiology of mental disorders have not been determined to date.

The postmortem brain study is essential for understanding the pathophysiology of the brain given that it is the only research method that allows the use of the brain tissue obtained from patients. Additionally, pathological changes, such as those related to astrocyte and dendritic cell spine densities, are identified in the postmortem brain of patients with mental disorders other than neurodegenerative diseases. ³ , ⁴ , ⁵ In contrast, the problems of mortality effects and age‐related changes in many cases make it difficult to identify changes specific to patients with mental disorders, and the results of the postmortem brain study only reflect the overall outcome of the lifelong effects of the disorders. Therefore, it is impossible to verify functional assessments and the pathogenesis of such disorders using this study.

In contrast, brain imaging studies can reveal the brain's construction and record its live activity in patients using methods such as functional magnetic resonance imaging (fMRI). For example, previous studies using brain imaging have reported abnormalities in the cortical‐cerebellar‐striatal‐thalamic loop in patients with SCZ and a significant decrease in striatal activation during reward feedback in patients with depression. ⁶ , ⁷ However, the spatiotemporal resolution used in brain imaging studies is insufficient to determine the molecular and cellular mechanisms; moreover, it is difficult to identify the details of the lesion and the dynamic course of mental disorders using these studies.

Animal models can be analyzed in a series of steps from molecular analysis to cellular, individual, and behavioral analyses, which are difficult to analyze by postmortem brain or brain imaging studies. Various animal models of mental disorders have been reported, such as zebrafish, mouse, rat, and marmoset models. ⁸ , ⁹ , ¹⁰ Among them, mouse models are most commonly used because of their genetic manipulation and ease of reproduction. Many studies have examined the pathogenesis of mental disorders using mouse models that recapitulate the genomic variants identified by genetic research as high risk factors for mental disorders. ¹¹ , ¹² Moreover, animal model studies mainly use experimental tools for drug development. However, these studies are often associated with several limitations, particularly the interspecies gap. Notably, the findings of mouse models do not necessarily translate to humans because of the differences between the mouse and human brains at the macro and micro levels. These differences include those related to the structure of the cerebral cortex (including the gyri or functional regions), cellular composition ratio, interactions between neurons and glial cells, gene expression in neurons, and expression patterns of serotonin receptors, which are one of the therapeutic targets for drugs for mental disorders. ¹³ , ¹⁴ , ¹⁵ Further evidence of these differences between humans and mice has been found in the cerebellum; for example, in the layered structure of early development, the spatial arrangement of progenitor cells, and the early loss of the rhombic lip in mice, which results in congenital cerebellar hypoplasia in humans. ¹⁶ , ¹⁷ , ¹⁸ Given these differences, it has been debated whether animal models accurately reflect all human changes.

Although the abovementioned approaches have provided important insights into mental disorders by compensating for each other's disadvantages, several factors are inevitably missing, and these include dynamic cellular and molecular phenotypes of human brain cells. Recently, human induced pluripotent stem cells (hiPSCs) have been considered a novel tool for the study of mental disorders. In particular, advances in iPSC technology have enabled the differentiation of iPSCs into neurons, astrocytes, oligodendrocytes, microglia, and various cells that make up the brain, facilitating analysis at the molecular, cellular, and functional levels using human cells. ¹⁹ , ²⁰ , ²¹ , ²² , ²³ , ²⁴ , ²⁵ , ²⁶ Additionally, organoid technology has been developed to differentiate into tissues using three‐dimensional (3D) structures that mimic in vivo organs, allowing for the analysis of cell–cell interactions and cell fate during early development. ²⁷ , ²⁸ , ²⁹ These advantages are expected to address the limitations of other approaches, such as those related to cellular and molecular phenotypes of living human brain cells/tissues, thereby helping in the determination of the pathogenesis and pathophysiology of mental disorders.

Research into mental disorders is at a major turning point. In particular, advances in various technologies, including iPSCs, provide significant new insights. This situation raises the following questions:

1. What is the benefit of using the combined use of iPSC technology and other tools for research into mental disorders?

2. How will the combined use of iPSC technology facilitate studies on the pathogenesis of mental disorders?

In the present review, we discuss the past and current trends, potential, limitation, and future of iPSC technologies in mental disorder research.

Trend History in iPSC‐Based Research on Mental Disorders

The establishment of mouse embryonic stem cells (ESCs), which could generate all types of mouse body cells, brought a new paradigm of creative science to generate several types of cells from ESCs. The generation of human ESCs allowed the application of this idea to the human system. ³⁰ , ³¹ In addition, their ability to generate virtually any cell type brought a paradigm shift in many fields of science. However, the establishment of ESCs involves the destruction of the embryo, occasionally raising ethical concerns about the related research. In 2006, a breakthrough technology was established by Takahashi and Yamanaka in which they established pluripotent stem cells from mouse epidermal cells by inducing only four factors, namely, Oct3/4, Sox2, Klf4, and c‐Myc. These cells were called iPSCs. Moreover, they established human iPSCs in 2007. Notably, iPSCs were associated with less ethical concerns and enabled the generation of human neuronal cells from both healthy control‐ and patient‐derived cells.

The researchers then applied iPSCs to the field of mental disorders. Initial studies of mental disorders using patient‐derived iPSCs focused on monogenic disorders, such as Rett syndrome (RTT). ³² The iPSC‐derived neurons from patients with RTT demonstrated fewer synapses, reduced spinal density, smaller soma size, altered calcium signaling, and electrophysiological defects compared with those from controls, thereby indicating the usefulness of iPSCs as a human experimental model. Since then, researchers in clinical neuroscience have used patient‐derived iPSCs to investigate the molecular and cellular pathogenesis of mental disorders.

One of the pioneering case‐control studies in mental disorders was conducted by Brennand et al. ³³ They found that the iPSC‐derived neurons of patients with SCZ had diminished neuronal connectivity as well as decreased neurite number, PSD95‐protein levels, and glutamate receptor expression. Subsequently, in other studies, an iPSC‐based approach was broadly applied to other mental disorders, such as ASD ³⁴ and bipolar disorder. ³⁵ These studies support that an iPSC‐based experimental model recapitulates the cellular and molecular signature of the donor patients.

Recent studies on patient‐derived iPSCs have been transferred to genetic variant‐based cohorts through the combined use of genomic information and iPSC technology because iPSC‐derived somatic cells can adopt the donor's genetic signature. Two technical breakthroughs may have helped reinforce this trend. The first is the advancement of omics technology, specifically transcriptomics technology. The widespread advances in transcriptome analysis have made it possible to analyze the comprehensive genome background of patient‐derived cells and determine the molecular changes in brain cells caused by the patient‐specific genetic variants. The second is the spread of CRISPR‐mediated genome editing technology. Although the efficiency of genome editing became much higher after the introduction of zinc finger nuclease and transcription activator‐like effector nuclease, its costs remained high, and it was almost impossible for most research laboratories to edit patient‐specific genetic variants. However, the integration of iPSCs into CRISPR technology allowed for a better and cheaper study of the importance of patient‐specific variants in the cells. Therefore, this tool has encouraged researchers to manipulate studies based on genetic variants in mental disorders. Furthermore, the two technical breakthroughs allow researchers to determine unknown genetic variants of the patient and manipulate the variant in patient‐derived cells, thereby accelerating the tailored analysis of diseases, including mental disorders.

Attempt to Understand the Genetic‐Based Latent Pathogenesis of Mental Disorders in a Dish

The study by Yoon et al. is a representative example of using patient‐derived iPSCs with a genetic risk variant. They provided a mechanistic understanding of how the detection of 15q11.2 deletion—a major risk factor for SCZ and ASD—affects neural developmental processes. ³⁶ Notably, they used human iPSCs as an entry point to identify targets associated with genetic variants, followed by the validation of in vivo physiological relevance. Their study was a milestone to indicate the potential of iPSCs in the genetic‐based pathogenesis of mental disorders.

Many studies, including ours, have used patient‐derived iPSCs with various genetic variants. ³⁷ , ³⁸ , ³⁹ , ⁴⁰ , ⁴¹ , ⁴² , ⁴³ , ⁴⁴ The addition of iPSCs to other approaches has widened the molecular and cellular phenotypes owing to genetic variants in humans. Here, we have discussed how the findings of other studies have been advanced using iPSCs considering 22q11.2 deletion as an example.

Patients with 22q11.2 deletion syndrome (22q11.2DS) exhibit various clinical presentations, such as congenital heart diseases, neuropsychiatric disorders, immunological problems, and cleft palate. The microdeletion of 22q11.2 is attributed to the presence of several large paralogous low copy repeats (LCR) containing a high degree of sequence identity (>97%), ⁴⁵ and it occurs in 1 out of 2000–4000 births. In particular, patients with 22q11.2DS are at a high risk of various neuropsychiatric disorders, such as intellectual disability, ASD, attention‐deficit/hyperactivity disorder, SCZ, epilepsy, and Parkinson's disease. Therefore, neuroscience researchers considered this deletion as an indication of the pathogenesis of neuropsychiatric disorders, including mental disorders. Notably, the study of 22q11.2DS offers the opportunity to open a way to unveil the brain pathophysiology underlying the onset of neuropsychiatric symptoms.

The animal models are well‐applied to elucidate the brain pathophysiology of 22q11.2DS. The mouse model of 22q11.2DS has provided important insights, including altered synaptic activities, ⁴⁶ impaired neural circuits, ⁴⁷ , ⁴⁸ and decreased auditory prepulse inhibition, ¹¹ which reflect the phenotypes of patients with 22q11.2DS. These findings highlight the biological impact of the deleted genes within the 22q11.2 region in a series of steps from the molecular to behavioral level. However, the chromosome 22‐specific LCRs (LCR22) are not detected in the mouse genome. ⁴⁹ In contrast, a similar structure can be detected in nonhuman primates; however, it is less complex in primates than in humans, and the LCR22 expansion is unique to the human population. ⁵⁰ Moreover, inevitable differences can be observed between the human and mouse brains at both micro and macro levels. Therefore, it can be suggested that there are also findings specific in humans with 22q11.2 deletion.

To address these issues, patient‐derived iPSCs have been used. The iPSC‐derived neuronal cells of patients with 22q11.2DS exhibit differentially expressed microRNAs owing to the deletion of DGCR8—one of the genes in the 22q11.2 region, ⁵¹ , ⁵² the propensity to differentiate into glial cells, ⁵¹ and mitochondrial deficits. ⁵³ These phenotypes have been found in other experimental models, such as animal models and postmortem study models, indicating the usefulness of patient‐derived iPSCs. In addition, a recent study by Khan et al. demonstrated defects in the neuronal activity of the 15 patients' iPSC‐derived neurons. ⁵⁴ A more recent study that used iPSCs derived from 20 patients with 22q11.2DS and genome‐edited iPSCs induced by 22q11.2 deletion found that the 22q11.2 deletion affected genes that converge with the psychiatric risk loci. ⁵⁵ Importantly, these cellular and molecular phenotypes were commonly observed in patients with 22q11.2DS, regardless of their clinical manifestations, such as ASD and SCZ. Thus, they may be the underlying or common neuronal phenotypes that cause the onset of various neuropsychiatric disorders.

As mentioned above, the analysis of patient‐derived iPSCs provided findings of the cellular and molecular phenotypes in the human neuronal cells with 22q11.2 deletion. However, the analysis of iPSCs of patients with 22q11.2DS mainly focused on the cortical neurons, with only a few findings about the other cellular types. Considering that 22q11.2 deletion is a cross‐disorder variant, findings in one cellular type are insufficient to determine the molecular/cellular features of neuronal cells in 22q11.2DS. To address this issue, we examined the phenotypes of another cellular type—the dopaminergic neurons. The next paragraph will introduce our study.

We generated iPSCs from healthy controls and patients with 22q11.2DS and then differentiated them into dopaminergic neurons. All iPSC lines differentiated into dopaminergic neurons with similar efficiency. We performed the semiquantitative proteomic analysis using mass spectrometry to examine the molecular alterations in dopaminergic neurons with 22q11.2DS. Overall, 272 differentially expressed proteins were identified in iPSC‐derived dopaminergic neurons from patients with 22q11.2DS compared to those derived from healthy controls. Kyoto Encyclopedia of Genes and Genomes pathway analysis using the differentially expressed proteins indicated that the protein processing in the endoplasmic reticulum (ER) is the most enriched pathway. Based on this proteomic analysis, we identified the dysfunction of protein kinase R‐like endoplasmic reticulum kinase (PERK) as a key molecule contributing to the cellular phenotypes in iPSC‐derived dopaminergic neurons of patients with 22q11.2DS; for example, it causes poor tolerance to ER stress, poor contact between the ER and mitochondria, and abnormal F‐actin dynamics. ³⁸ , ⁵⁶ These phenotypes are detected across subjects and lines, regardless of their clinical manifestations. In addition, the mouse model of 22q11.2DS shows reduced expression of the PERK protein, but slightly less reduced than expression in iPSC‐derived neurons from the patients. Although we used the iPSCs derived from small cohorts (n = 3), it can be assumed that the PERK dysfunction‐dependent phenotypes are a part of the human‐specific pathogenesis of 22q11.2DS. Owing to the use of patient‐derived iPSCs, we found new clues to determine the pathogenesis of 22q11.2DS. We believe that this approach will shed light on the latent pathogenesis of mental disorders.

Briefly, iPSCs with genetic risk variants have the potential to expand findings in the current literature. In addition, studies examining the shared molecular and cellular phenotypes of different risk variants have been recently reported. For example, neurons generated from patient iPSCs with RELN and PCDH15 deletions shared characteristics of shorter dendrites and fewer synapses. ⁵⁷ Furthermore, an iPSC‐derived brain organoid with three distinct genetic variants (i.e., SUV420H1, ARID1B, and CHD‐8) associated with ASD showed convergency in the asynchronous development of GABAergic neurons and deep‐layer excitatory projection neurons, ⁵⁸ probably offering answers to the question of “Why different genetic variants exhibit same clinical phenotypes in mental disorders?” According to these findings, iPSC‐based genetic studies will increasingly consider the diversity of genetic variants, rather than just one variant at a time, associated with mental disorders.

Potential of Organoid Technology for Mental Disorder Research

Historical overview of 3D brain tissue generation from pluripotent stem cells (PSCs)

Because PSCs have the potential to differentiate into all types of body cells, it has become possible to produce organ‐like structures in a dish owing to the emergence of PSCs. ⁵⁹ , ⁶⁰ , ⁶¹ Organoids are defined as 3D tissues that recapitulate cellular component and tissue structures, including in vivo developmental dynamics. ⁶² , ⁶³ The human 3D cerebral structure, which is currently considered as the pioneering work of cerebral organoid, was first generated in 2008 by Eiraku et al. ⁶⁴ They also conducted a follow‐up study demonstrating enhanced brain tissue formation, recapitulating up to the early second trimester of human brain development in 2013. ⁶⁵ In the same year, Jürgen Knoblich's group also produced a 3D human brain tissue ⁶⁶ and called its structures cerebral organoids. In particular, they were able to model microcephaly using patient‐derived iPSCs, paving the way for modeling neurological disorders through cerebral organoid technology. The Zika virus outbreak in South America around 2015 was an early example of the use of brain organoids as a disease modeling tool. As Zika virus infection during pregnancy is associated with congenital disabilities (including microcephaly), several studies have used cerebral organoids to confirm the pathogenesis of microcephaly caused by Zika virus infection by creating disease models via the direct infection of the virus into human ESC and iPSC‐derived cerebral organoids; moreover, they identified candidates for therapeutic drugs, such as TLR3 inhibitor, ivermectin, and Duramycine. ⁶⁷ , ⁶⁸ , ⁶⁹ , ⁷⁰ , ⁷¹ These studies made organoid technologies popular through the application of cerebral organoids.

To date, various region‐specific brain organoids have been generated, including the cerebral cortex, hypothalamus, ventral telencephalon, optic cup, anterior pituitary, cerebellum, hippocampus, choroid plexus, thalamus, and spinal cord. ⁶⁵ , ⁷² , ⁷³ , ⁷⁴ , ⁷⁵ , ⁷⁶ , ⁷⁷ , ⁷⁸ , ⁷⁹ , ⁸⁰ , ⁸¹ , ⁸² , ⁸³ , ⁸⁴ , ⁸⁵ , ⁸⁶ , ⁸⁷

Future perspectives for recapitulating latent pathogenesis of mental disorders using neural organoids

By providing living 3D structured human neural tissues, neural organoids offer unprecedented opportunities to study human neurodevelopment and disease. Although commonly used animal models have shown favorable results, some specific characteristics of the human brain, such as the existence of many outer radial glial cells, cannot be recapitulated in evolutionarily ancient species. The human neuronal monolayer culture is valuable for generating homogeneous neural cells, such as dopaminergic and motor neurons of the spinal cord. However, it cannot fully mimic the 3D structure and neural network between different neural regions. Human stem cell‐derived organoids can overcome the aforementioned limitations of monolayer neuronal culture by providing 3D neural structures with human‐specific characteristics. Furthermore, connections between the different regions become feasible by combining several region‐specific organoids. ⁸⁸ , ⁸⁹ Thus, as an accessible human 3D brain surrogate, neural organoids can provide a better platform to study neuropsychiatric disorders.

Furthermore, regionalized neural organoids can be used to facilitate research in the field of mental disorders. In particular, several areas of the brain of the patient with 22q11.2DS are known to be affected, such as the cerebral cortex, hippocampus, midbrain, and cerebellar. By recapitulating the developmental process of each region using patient‐derived iPSCs, the hidden mechanisms of each pathology could become accessible. Moreover, researchers will be able to analyze the pathogenesis of each region, which may lead to several symptoms in the patient. In addition, the use of living materials makes it possible to access the functional pathophysiology of mental disorders. For example, researchers can measure spontaneous neural activities, network activity patterns, and drug response of each region of the neural organoids via calcium imaging or electrophysiological studies.

In addition, xenotransplantation of organoids into animal brains will open additional avenues for their application among researchers. For example, it is known that transplantation of cerebral organoids into the animal brain promotes vascularization from the host brain and results in better differentiation of neurons, ⁹⁰ , ⁹¹ better proliferation, ⁹² axonal extension along with host neural circuit, ⁹³ , ⁹⁴ and modulation of the behaviors of transplanted animals. ⁹¹ , ⁹³ Furthermore, by inducing more matured neurons from patient‐derived iPSCs by transplantation, modeling of mental disorders, such as Timothy syndrome, will also be promoted. ⁹¹

Thus, neural organoid technologies have an extraordinary potential to facilitate studies on the latent aspects of mental disorders.

Limitations

Reproducibility is one of the major limitations of iPSC technology. The variation in an iPSC‐based disease model is attributed to differences in donor individuals and genetic stability, affecting differentiation potential, cellular heterogeneity, morphology, and the quantity of transcripts and proteins. ⁹⁵ Nongenetic variables may cause significant laboratory variance even when using a standardized experimental procedure. ⁹⁶ This issue should be resolved to advance to clinical application because it may cause inconsistent reproducibility of neural induction.

Another issue is the maturity of cells/tissues produced from iPSC. Owing to the limitations of the current culture technology, the cells/tissues (such as neurons and cerebral organoids) produced from iPSCs are not mature enough. Moreover, their gene expression resembles that of the fetal brain up to the second trimester, ⁹⁷ , ⁹⁸ , ⁹⁹ which may affect the interpretation of data regarding the stage of development. With the use of these properties of iPSC‐derived neurons and brain organoids, it has been found that pathophysiology in patients with mental disorders begins to emerge from developing brains. ¹⁰⁰ , ¹⁰¹ However, it is difficult to determine how the phenotypic variations in iPSC‐derived cells/tissues, which represent the earliest stages of human neurodevelopment, are related to the patient's pathophysiology at their mature stage. Certainly, further studies are needed to determine whether the iPSC‐based findings are related to the causal factors of the clinical symptoms.

Further, the accuracy of cerebral organoid technology still needs to be evaluated and improved to accurately model human brain development. According to a recent single‐cell RNA sequencing study of cerebral organoids, ¹⁰² iPSC‐derived brain organoids have lower cell‐type fidelity than primary samples.

Owing to these circumstances, iPSCs will not completely replace current methods but rather fill the gap in our understanding of the pathogenesis and pathophysiology of mental disorders. These limitations remain as open questions for researchers to explore the next generation of iPSC research fields.

Conclusion

Based on the results of recent studies on mental disorders, the use of only one approach is not effective enough to determine the pathogenesis and pathophysiology of mental disorders. The Research Domain Criteria by NIMH suggest that the next generation of research on mental disorders will require studies across multiple areas of analysis, including genes, neural circuits, and behavior. The use of iPSC technology will shed light on the human molecular and cellular mechanisms between genetic variants and clinical phenotypes in mental disorders (Fig. 2). Despite the limitations of iPSC research, iPSC technology provides a novel approach in the classification of the latent mechanisms of psychiatric symptoms by providing neuronal cells/tissues with disease‐related living material. Further, the field of mental disorder research will continue to grow to develop novel treatment approaches for mental disorders.

Fig. 2.

The use of iPSC technology based on genetic information to determine the molecular and cellular pathogenesis and pathophysiology of mental disorders.

Disclosure statement

HS was funded by Otsuka Pharmaceutical Company. NO has received research support or speakers' honoraria from, or has served as a joint researcher with, or a consultant to, Sumitomo Pharma, Eisai, Otsuka, KAITEKI, Mitsubishi Tanabe, Shionogi, Eli Lilly, Mochida, DAIICHI SANKYO, TSUMURA, Takeda, Meiji Seika Pharma, Kyowa, EA Pharma, Viatris, Kyowa Kirin, MSD, Janssen, Yoshitomi, Ricoh, Taisho, Nippon Boehringer Ingelheim, outside the submitted work. YA and HO declare no conflict of interest.

Author contributions

All authors wrote, reviewed, edited, and approved the manuscript.

Data Availability Statement

There are no shared data.