Background: The development of induced pluripotent stem cells (iPSCs) technology has enabled human cellular disease modeling for inaccessible cell types, such as neural cells in the brain. However, many of the iPSC-derived disease models established to date typically involve only a single cell type. These monoculture models are inadequate for accurately simulating the brain environment, where multiple cell types interact. The limited cell type diversity in monoculture models hinders the accurate recapitulation of disease phenotypes resulting from interactions between different cell types. Therefore, our goal was to create cell models that include multiple interacting cell types to better recapitulate disease phenotypes. Methods: To establish a co-culture model of neurons and astrocytes, we individually induced neurons and astrocytes from the same iPSCs using our novel differentiation methods, and then co-cultured them. We evaluated the effects of co-culture on neurons and astrocytes using immunocytochemistry, immuno-electron microscopy, and Ca²⁺ imaging. We also developed a co-culture model using iPSCs from a patient with familial Alzheimer's disease (AD) patient (APP^V717L mutation) to investigate whether this model would manifest disease phenotypes not seen in the monoculture models. Results: The co-culture of the neurons and astrocytes increased the branching of astrocyte processes, the number of GFAP-positive cells, neuronal activities, the number of synapses, and the density of presynaptic vesicles. In addition, immuno-electron microscopy confirmed the formation of a tripartite synaptic structure in the co-culture model, and inhibition of glutamate transporters increased neuronal activity. Compared to the co-culture model of the control iPSCs, the co-culture model of familial AD developed astrogliosis-like phenotype, which was not observed in the monoculture model of astrocytes. Conclusions: Co-culture of iPSC-derived neurons and astrocytes enhanced the morphological changes mimicking the in vivo condition of both cell types. The formation of the functional tripartite synaptic structures in the co-culture model suggested the mutual interaction between the cells. Furthermore, the co-culture model with the APP^V717L mutation expressed in neurons exhibited an astrocytic phenotype reminiscent of AD brain pathology. These results suggest that our co-culture model is a valuable tool for disease modeling of neurodegenerative diseases.

Alzheimer’s disease (AD) is an aging-dependent neurodegenerative disease that impairs cognitive function. Although the main pathologies of AD are the aggregation of amyloid-beta (Aβ) and phosphorylated Tau protein, the mechanisms that lead to these pathologies and their effects are believed to be heterogeneous among patients. Many epidemiological studies have suggested that sex is involved in disease prevalence and progression. The reduction of sex hormones contributes to the pathogenesis of AD, especially in females, suggesting that the supplementation of sex hormones could be a therapeutic intervention for AD. However, interventional studies have revealed that hormone therapy is beneficial under limited conditions in certain populations with specific administration methods. Thus, this suggests the importance of identifying crucial factors that determine hormonal effects in patients with AD. Based on these factors, it is necessary to decide which patients will receive the intervention before starting it. However, the long observational period and many uncontrollable environmental factors in clinical trials made it difficult to identify such factors, except for the APOE ε4 allele. Induced pluripotent stem cells (iPSCs) derived from patients can differentiate into neurons and recapitulate some aspects of AD pathogenesis. This in vitro model allows us to control non-cell autonomous factors, including the amount of Aβ aggregates and sex hormones. Hence, iPSCs provide opportunities to investigate sex-dependent pathogenesis and predict a suitable population for clinical trials of hormone treatment.

Sporadic Alzheimer's disease (sAD) is a neurodegenerative disease that has the highest prevalence among patients with dementia. The genetic risk factors for sAD are comprised of many single nucleotide polymorphisms (SNPs), as indicated by genome-wide association studies. Herein, we generated the KEIOi005-A-induced pluripotent stem cell (iPSC) line from urine-derived cells (UDCs) of a mild Alzheimer's disease (AD) patient with multiple sAD risk SNPs comprising T > C heterozygous APOE ε3/ε4 (rs429358), A > G heterozygous BIN1 (rs744373), and T > G homozygous MS4A6A (rs610932). The established iPSC line demonstrates normal stemness and pluripotency and can be used for in vitro disease modeling of AD.