Sopak Supakul

ABSTRACT Several protocols for generating oligodendrocytes (OLs) from human pluripotent stem cells have been reported. However, they are limited by long culture duration, intensive handling, and low yield of mature OLs. Transcription factor‐based strategies have improved efficiency, but OLIG2 and SOX10, key regulators of oligodendrocyte precursor cells (OPCs), also promote alternative neural fates. Here, we developed a Tet‐inducible system to control SOX10 and OLIG2 expression, including that of a phosphorylation‐deficient OLIG2 mutant (S147A). Co‐expression of SOX10 and OLIG2 enhanced OPC induction, confirmed by O4 positivity and transcriptomic profiling. Interestingly, only a brief induction of SOX10 + OLIG2(S147A) (2–5 days) efficiently yielded myelin basic protein positive OLs within 25 days, reaching approximately 20% of total cells. In contrast, sustained doxycycline‐mediated expression of SOX10 and OLIG2(S147A) favored OPC proliferation and delayed OL maturation. These findings highlight the importance of temporal control of transcription factor activity in accelerating OL differentiation and provide a practical platform for disease modeling and regenerative applications.

Amyotrophic Lateral Sclerosis/Parkinsonism-Dementia Complex (ALS/PDC), a rare and complex neurological disorder, is predominantly observed in the Western Pacific islands, including regions of Japan, Guam, and Papua. This enigmatic condition continues to capture medical attention due to affected patients displaying symptoms that parallel those seen in either classical amyotrophic lateral sclerosis (ALS) or Parkinson's disease (PD). Distinctly, postmortem examinations of the brains of affected individuals have shown the presence of α-synuclein aggregates and TDP-43, which are hallmarks of PD and classical ALS, respectively. These observations are further complicated by the detection of phosphorylated tau, accentuating the multifaceted proteinopathic nature of ALS/PDC. The etiological foundations of this disease remain undetermined, and genetic investigations have yet to provide conclusive answers. However, emerging evidence has implicated the contribution of astrocytes, pivotal cells for maintaining brain health, to neurodegenerative onset, and likely to play a significant role in the pathogenesis of ALS/PDC. Leveraging advanced induced pluripotent stem cell technology, our team cultivated multiple astrocyte lines to further investigate the Japanese variant of ALS/PDC (Kii ALS/PDC). CHCHD2 emerged as a significantly dysregulated gene when disease astrocytes were compared to healthy controls. Our analyses also revealed imbalances in the activation of specific pathways: those associated with astrocytic cilium dysfunction, known to be involved in neurodegeneration, and those related to major neurological disorders, including classical ALS and PD. Further in-depth examinations revealed abnormalities in the mitochondrial morphology and metabolic processes of the affected astrocytes. A particularly striking observation was the reduced expression of CHCHD2 in the spinal cord, motor cortex, and oculomotor nuclei of patients with Kii ALS/PDC. In summary, our findings suggest a potential reduction in the support Kii ALS/PDC astrocytes provide to neurons, emphasizing the need to explore the role of CHCHD2 in maintaining mitochondrial health and its implications for the disease.

Abstract 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.