Otomo Asako, Nishijima Keiko, Murakami Yuta, Ishikawa Mitsuru, Yudahira Haruka, Shimakura Kento, Okano Hideyuki, Aoki Masashi, Kimura Hiroshi, Hadano Shinji
Molecular Neuropathobiology Laboratory, Department of Physiology, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
Micro/Nano Technology Center, Tokai University, Hiratsuka, Kanagawa, Japan.
Front Cell Neurosci. 2025 Jul 24;19:1590732. doi: 10.3389/fncel.2025.1590732. eCollection 2025.
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease caused by the loss of upper and lower motor neurons. Mutations in the FUS/TLS gene have been reported as the second most common mutation in Japanese patients with familial ALS. In recent years, lower motor neurons (LMNs) differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients have been widely used to analyze the mechanisms of neuronal cell death and degeneration.
In this study, we developed a microfluidic device designed to observe axonal growth, morphology, and trafficking at high resolution in neurons derived from induced pluripotent stem cells (iPSCs) and tested whether our microfluidic device effectively evaluates neurodegenerative phenotypes. We used iPSCs carrying homozygous FUS/TLS mutations (FUS_H517D) to induce LMNs by expressing NEUROG2, ISL1, and LHX3 under the control of the tetracycline regulation system.
After seven days of in vitro differentiation (DIV7), we confirmed that over 95% of iPSCs differentiated into HB9-positive LMNs. Notably, the cell viability of FUS_H517D LMNs was comparable to that of LMNs differentiated from iPSCs without the FUS/TLS mutation at DIV7. However, by DIV14 and DIV21, the viability of FUS_H517D LMNs was notably lower than that of control LMNs, indicating degeneration of FUS_H517D LMNs after differentiation. Using our microfluidic device, we assessed axonal phenotypes in FUS_H517D LMNs. Under oxidative stress conditions, we observed that the axonal length of FUS_H517D LMNs was significantly shorter than that of control cells as early as DIV7, with this axonal growth restriction becoming more pronounced by DIV11. This suggests that axonal growth restriction is an early detectable phenotype in degenerating neurons. Additionally, we examined mitochondrial trafficking within axons in our device, which is often disrupted in degenerative neurons. Our results showed a significant increase in the number of motile mitochondria in FUS_H517D LMNs, with retrograde transport accounting for a large portion of trafficking. Our microfluidic device-based culture and evaluation system using FUS_H517D LMNs offers a valuable ALS cellular model focused on early axonal phenotypes. This approach contributes to the study of molecular mechanisms underlying axonal degeneration in ALS.
肌萎缩侧索硬化症（ALS）是一种由上下运动神经元丧失引起的进行性神经退行性疾病。据报道，FUS/TLS基因突变是日本家族性ALS患者中第二常见的突变。近年来，源自ALS患者的诱导多能干细胞（iPSC）分化而来的下运动神经元（LMN）已被广泛用于分析神经元细胞死亡和退化的机制。
在本研究中，我们开发了一种微流控装置，旨在高分辨率观察源自诱导多能干细胞（iPSC）的神经元的轴突生长、形态和运输，并测试我们的微流控装置是否能有效评估神经退行性表型。我们使用携带纯合FUS/TLS突变（FUS_H517D）的iPSC，通过在四环素调控系统的控制下表达NEUROG2、ISL1和LHX3来诱导LMN。
体外分化7天后（DIV7），我们证实超过95%的iPSC分化为HB9阳性的LMN。值得注意的是，FUS_H517D LMN在DIV7时的细胞活力与未发生FUS/TLS突变的iPSC分化而来的LMN相当。然而，到DIV14和DIV21时，FUS_H517D LMN的活力明显低于对照LMN，表明分化后FUS_H517D LMN发生了退化。使用我们的微流控装置，我们评估了FUS_H517D LMN的轴突表型。在氧化应激条件下，我们观察到早在DIV7时，FUS_H517D LMN的轴突长度就明显短于对照细胞，到DIV11时这种轴突生长受限更加明显。这表明轴突生长受限是退化神经元中早期可检测到的表型。此外，我们检查了我们装置中轴突内的线粒体运输情况，这在退化神经元中经常受到干扰。我们的结果显示FUS_H517D LMN中活动线粒体的数量显著增加，逆行运输占运输的很大一部分。我们基于微流控装置的使用FUS_H517D LMN的培养和评估系统提供了一个有价值的专注于早期轴突表型的ALS细胞模型。这种方法有助于研究ALS中轴突退化的分子机制。
