INTRODUCTION
Recent advancements in the field of induced pluripotent stem cell (iPSC) research have shown promise, helping to address the complexities of neurodegenerative diseases. Since iPSCs were first introduced by the Yamanaka research team [1], this technology has been applied across numerous fields, revolutionizing various aspects of biomedical research and clinical practice. iPSCs are created from adult somatic cells that have been reprogrammed to an embryonic-like pluripotent state through the introduction of specific transcription factors, including SRY-box transcription factor 2 (Sox2), octamer binding transcription factor 4 (Oct4), KLF4, and c-myc [1]. More recently, iPSCs have been applied in both disease modeling and personalized medicine. Using iPSC technology, specific cell types of interest can be generated from a patient’s somatic cells, allowing for the creation of patient-specific drug testing models.
Neurodegenerative diseases encompass a diverse group of disorders characterized by progressive degeneration of the central nervous system, leading to neuronal loss and impaired glial cell function. Neurodegenerative diseases result in significant impairment of cognitive and motor functions. The application of iPSC technology in the research of neurodegenerative diseases has several advantages. First, the conventional approach for studying neurodegenerative diseases relies heavily on animal models, which show significant physiological differences with humans. Unlike animal models, iPSC-based disease models offer a more accurate context for investigating the complex pathophysiology of human neurodegenerative conditions. Second, iPSCs can be derived relatively easily from a patient’s fibroblasts or peripheral blood mononuclear cells, whereas human brain tissue samples can only be obtained post-mortem. Third, patient-derived iPSCs retain mutant variants of disease-causing genes, possess the properties of embryonic stem cells that can be differentiated into disease-specific brain cells, including neurons, glia, astrocytes, and oligodendrocytes, enabling the investigation of disease pathology in vitro.
Although dementia is a common phenotype of neurodegenerative diseases, rare forms of dementia are caused by single-gene mutations. Understanding the genetic basis and pathomechanism of dementia arising from single-gene mutations is crucial for expanding our knowledge in the field of dementia research. Familial Alzheimer’s disease (AD) accounts for less than 1% of all AD cases. Mutations in the presenilin-1 (PSEN1), presenilin-2 (PSEN2), or amyloid precursor protein (APP) genes lead to the onset of familial AD; however, the exact mechanism of these genes are still being explored. Recent advances in iPSC models have paved the way for investigating the role of these genes in the pathogenesis of AD. Research using iPSC technologies has also facilitated our understanding of rare leukodystrophies, such as Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), caused by mutations in the notch receptor 3 (NOTCH3) gene; and adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), caused by mutations in the colony-stimulating factor 1 receptor (CSF1R) gene.
This review focuses on summarizing the current state of iPSC-based research, particularly in modeling dementia caused by single-gene mutations, highlighting the extent of current investigations and their implications. Furthermore, we summarize existing studies incorporating organoids, three-dimensional (3D) culture systems, drug screening, and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology. By examining these advanced methodologies, this review aimed to delineate and propose future research directions for rare neurodegenerative diseases attributed to specific gene mutations.
ALZHEIMER’S DISEASE
PSEN1, PSEN2, APP
AD is the most common neurodegenerative disease worldwide. Familial AD is characterized by the early onset of symptoms, caused by mutations in PSEN1, PSEN2, or APP genes. These mutations can increase the production of amyloid-beta (Aβ) peptides, particularly the aggregation-prone Aβ42, contributing to amyloid plaque formation.
The existing studies using iPSC technology to enhance our understanding of AD are summarized in Table 1. To investigate the effect of these causative genes on Aβ mechanistic progress, iPSC technology has been utilized to examine AD pathology in various type of brain cells, including neurons, microglia, and astrocytes [2-5]. Studies using iPSC-derived neurons (iN) and neural progenitor cells (iNPC) with APP and PSEN1 mutations have reported increased production of β-amyloid and a higher Aβ42/40 ratio compared to normal cell lines, thus confirming the modeling of AD [2,5,6]. Regarding the metabolic impact of PSEN1 ∆E9 mutation in AD, studies using iPSC-derived astrocytes revealed pathological hallmarks and changes in neuron-supportive functions, including increased oxidative stress and reduced lactate secretion [3]. In addition, iPSC-derived basal forebrain cholinergic neurons (BFCNs) harboring PSEN2 N141I mutation have been utilized to explore the mechanisms underlying short-term memory formation in AD, revealing electrophysiological deficits in BFCNs from AD lines [4]. Further, to delve deeper on memory formation within APP mutation, Pomeshchik et al. [7] generated iPSC-derived hippocampal spheroids, subsequently uncovering a reduction in synaptic proteins and an increased Aβ 42/40 peptide ratio, thereby elucidating the mechanisms underlying memory deficits in AD. Furthermore, to better mimic the complexity of the human brain, further studies have reported the successful modeling of 3D culture cerebral organoids for sporadic AD, as well as those carrying PSEN1 mutations [8-10].
iPSCs have also been leveraged as a research tool in bioinformatics research. For example, alterations in gene expression have been identified through comprehensive molecular profiling and analysis of single-cell transcriptomes in cells carrying PSEN1 and APP mutations [11,12]. For drug screening, iPSC-derived cortical neurons carrying the G384A mutation in the PSEN1 gene were used to screen 1,000 pharmaceutical compounds, revealing 27 compounds which reduced Aβ load [13]. Another drug screening study using iNs revealed several compounds that target cholesterol metabolism, thereby reducing phospho-Tau accumulation, suggesting therapeutic potential for AD [14].
Aside from studies on rare familial AD-causing genes, research on sporadic AD has aimed to understand the mechanisms of apolipoprotein E4 (APOE4), which is the single greatest risk factor for AD. Using CRISPR/Cas9 engineering, isogenic paired cell lines have been generated to investigate how APOE4 contributes to the impairment of synaptic function, lipid metabolism, and immune responses in iNs, astrocytes, and microglial cells [15-17].
CADASIL
CADASIL is a rare genetic small-vessel disease resulting from specific inherited variants of the NOTCH3 gene [18]. Individuals with CADASIL commonly experience recurrent strokes that can lead to cognitive impairment and vascular dementia [19]. The pathological changes observed in CADASIL, including vascular smooth muscle cell (VSMC) degeneration and accumulation of the NOTCH3 protein, all contribute to vascular dysfunction through the deposition of granular osmiophilic materials around VSMCs [20,21]. The central nervous system has a distinct structure, known as the neurovascular unit, that facilitates the functional connections between blood vessels and neurons. This unit primarily comprises brain microvascular endothelial cells (BMEC), mural cells, astrocytes, and neurons [22]. These cells work in concert to regulate the blood-brain barrier (BBB) function and cerebral blood flow, thereby ensuring homeostasis of the central nervous system [23].
To understand the role of NOTCH3 mutations in the neurovascular unit of CADASIL, various in vitro studies have been conducted to observe the pathological changes in multiple cell types derived from iPSCs [24,25]. For example, Zhang et al. [24] generated an iPSC-derived neurovascular unit model including BMECs, astrocytes, and cortical projection neurons, and co-cultured each cell type in a transwell to mimic the BBB in order to investigate the effect of the NOTCH3 mutant on neurovascular interactions and BBB function. Zhang et al. [24] found impaired electrical resistance in mural cells and astrocytes, along with significantly decreased barrier function and disorganized tight junctions in BMECs in the NOTCH3 mutant neurovascular unit model. Another study further showed that mural cells derived from iPSCs carrying the NOTCH3 mutation (R182C, R141C, C106R) exhibited increased platelet-derived growth factor receptor β (PDGFRβ) expression, abnormal structure and distribution of the filamentous actin network, and the Notch3 extracellular domain (N3ECD)/latent-transforming growth factor β-binding protein-1 (LTBP-1)/high temperature requirement A1 (HtrA1)-immunopositive deposits, thus recapitulating pathogenic changes of CADASIL [25]. In another CADASIL research, alterations in gene expression within the NOTCH and nuclear factor κB signaling pathways, as well as cytoskeleton disorganization were observed in VSMCs which were differentiated from patient-derived iPSCs [26].
ALSP
ALSP is a rare, autosomal dominant, white matter degenerative disorder characterized by cognitive impairment arising from mutations in the CSF1R gene [29]. CSF1R is a tyrosine kinase receptor, and approximately 75% of ALSP-CSF1R mutations are located in the tyrosine kinase domain [30]. CSF1R plays a vital role in the proliferation and survival of microglia, as well as in the differentiation of neural progenitor cells [30]. This led to the hypothesis that individuals with CSF1R mutations associated with ALSP may display imbalanced microglial function and impaired neuronal activity.
Since CSF1R mutations impair microglial proliferation and survival, the generation of an in vitro model of patient-derived iPSC differentiation into microglia harboring CSF1R mutations is challenging. Previous studies have generally used CSF1R haploinsufficient (Csf1r+/-) mouse models or zebrafish models with CSF1R mutations to investigate the mechanistic relationship between CSF1R and microglia [31,32]. However, despite these efforts, the exact mechanisms linking ALSP and CSF1R remains elusive, partly because of species differences as mouse or zebrafish models are different from humans.
Researchers have generated ALSP-iPSC disease models from fibroblasts of ALSP patients [33] or by using the CRISPR/Cas9-based CSF1R knockout technology from iPSC lines of normal human [34]. However, as CSF1R gene is critical in the differentiation of microglia and neural progenitor cells, there was a hurdle to differentiate ALSP-iPSCs into these cell types. A recently reported novel protocol allows for more efficient differentiation of iPSCs from ALSP patients with CSF1R mutations into microglia [35]. Using this novel protocol, the differentiated ALSP-induced microglia transcriptomic analysis revealed a gene expression pattern similar to that of human microglia [35]. Moreover, these cells display characteristics of ALSP pathology, including decreased CSF1R autophosphorylation, impaired migratory ability, and reduced purinergic receptor P2Y12 (P2RY12) expression [35].
Following the successful establishment of ALSP-induced microglial lines carrying CSF1R mutations, future studies should focus on exploring the mechanistic correlation between CSF1R and microglia in a human-centric manner. This could be achieved by investigating gene-corrected isogenic lines using CRISPR/Cas9 technology. Furthermore, investigating the phenotype in 3D cultures using a co-culture system of neurons and non-neuronal cells [36] or organoids [37,38] will be crucial in understanding the complex interactions within the brain microenvironment. Additionally, leveraging insights from research on microglial phenotypes in other diseases, and applying them to ALSP pathology, will help enhance our understanding of this disease.
APPLICATIONS
iPSCs have diverse applications, ranging from differentiation into disease-specific cell types, to exploration of the mechanisms using gene-editing technologies in cell models. Additionally, iPSCs have been actively studied for their potential in expanding into 3D culturable organoids, cell replacement therapy, and drug screening. For example, cell replacement therapy involving the injection of iPSC-derived dopamine progenitor cells into a humanized mouse model, has been investigated in Parkinson’s disease, another common neurodegenerative condition [39]. iPSC disease modeling can further be used for drug screening, facilitating not only high-throughput drug discovery but also the development of personalized medicine tailored to individual symptoms, in vivo safety and efficacy testing of developed drugs, and examination of host-microbe interactions [40]. Additionally, gene-editing technologies are expected to be increasingly used in therapeutic studies owing to continuous advancements such as Cas12f, which reduces off-target effects and enhances delivery [41]. These advancements further highlight the critical role of iPSCs in understanding disease pathology and developing therapeutic strategies. The importance of iPSC research in the field of rare diseases is expected to increase, offering new insights and treatment avenues.
FUTURE DIRECTIONS
Although iPSCs have significant potential in the field of rare neurodegenerative diseases, there are some limitations that should be considered in future research. Owing to their cellular reprogramming to the pluripotent state, iPSCs exhibit rejuvenated epigenetic features, including DNA and cytosine-phosphate-guanine (CpG) methylation, which are both signatures of aging [42]. To overcome this issue, research is currently underway on aging induction by directly reprogramming somatic cells to iNs [43], or by treating iPSC-derived cells with chemicals such as telomerase inhibitors [44]. Further advancements in gene-editing technologies for iPSCs have raised concerns regarding off-target effects and genomic instability [45,46]. Despite technological advancements, iPSCs still face limitations in accurately replicating the mechanical structure and environmental conditions of human organs in dishes, even with the development of 3D-culturable organoids [47-49]. Furthermore, while iPSCs offer the advantage of autologous transplantation [45], their immortalized nature raises concerns regarding issues such as cell engraftment, immunogenicity, and tumorigenicity, which must be carefully addressed to ensure their safety and efficacy [50-52]. As such, a comprehensive understanding of the molecular characteristics of these models is essential to harness their full potential in the context of neurodegenerative diseases.
CONCLUSION
Research utilizing iPSC-based disease modeling has made it feasible to investigate disease phenotypes and their underlying mechanisms in rare neurodegenerative diseases caused by single-gene mutations. Various iPSC-derived brain cell types have successfully replicated the phenotypes of familial AD, CADASIL, and ALSP. Additionally, many studies have employed gene editing to create isogenic iPSC lines for the exploration of pathogenic pathways. These findings underscore the significance of iPSC-based research in advancing our understanding of rare neurodegenerative diseases.
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