Investigation of early axonal phenotypes in an iPSC-derived ALS cellular model using a microfluidic device
IntroductionAmyotrophic 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 neu...
Saved in:
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2025-07-01
|
| Series: | Frontiers in Cellular Neuroscience |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fncel.2025.1590732/full |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849709349007523840 |
|---|---|
| author | Asako Otomo Asako Otomo Keiko Nishijima Yuta Murakami Mitsuru Ishikawa Mitsuru Ishikawa Haruka Yudahira Kento Shimakura Hideyuki Okano Hideyuki Okano Masashi Aoki Hiroshi Kimura Hiroshi Kimura Shinji Hadano Shinji Hadano |
| author_facet | Asako Otomo Asako Otomo Keiko Nishijima Yuta Murakami Mitsuru Ishikawa Mitsuru Ishikawa Haruka Yudahira Kento Shimakura Hideyuki Okano Hideyuki Okano Masashi Aoki Hiroshi Kimura Hiroshi Kimura Shinji Hadano Shinji Hadano |
| author_sort | Asako Otomo |
| collection | DOAJ |
| description | IntroductionAmyotrophic 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.MethodsIn 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.Results and discussionsAfter 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. |
| format | Article |
| id | doaj-art-d0cde4e522904fe28f803738824e031c |
| institution | DOAJ |
| issn | 1662-5102 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cellular Neuroscience |
| spelling | doaj-art-d0cde4e522904fe28f803738824e031c2025-08-20T03:15:20ZengFrontiers Media S.A.Frontiers in Cellular Neuroscience1662-51022025-07-011910.3389/fncel.2025.15907321590732Investigation of early axonal phenotypes in an iPSC-derived ALS cellular model using a microfluidic deviceAsako Otomo0Asako Otomo1Keiko Nishijima2Yuta Murakami3Mitsuru Ishikawa4Mitsuru Ishikawa5Haruka Yudahira6Kento Shimakura7Hideyuki Okano8Hideyuki Okano9Masashi Aoki10Hiroshi Kimura11Hiroshi Kimura12Shinji Hadano13Shinji Hadano14Molecular Neuropathobiology Laboratory, Department of Physiology, Tokai University School of Medicine, Isehara, Kanagawa, JapanMicro/Nano Technology Center, Tokai University, Hiratsuka, Kanagawa, JapanMolecular Neuropathobiology Laboratory, Department of Physiology, Tokai University School of Medicine, Isehara, Kanagawa, JapanDepartment of Mechanical Engineering, Tokai University School of Engineering, Hiratsuka, Kanagawa, JapanDivision of CNS Regeneration and Drug Discovery, International Center for Brain Science, Fujita Health University, Aichi, JapanDepartment of Physiology, Keio University School of Medicine, Tokyo, JapanMolecular Neuropathobiology Laboratory, Department of Physiology, Tokai University School of Medicine, Isehara, Kanagawa, JapanMolecular Neuropathobiology Laboratory, Department of Physiology, Tokai University School of Medicine, Isehara, Kanagawa, JapanDivision of CNS Regeneration and Drug Discovery, International Center for Brain Science, Fujita Health University, Aichi, JapanDepartment of Physiology, Keio University School of Medicine, Tokyo, JapanDepartment of Neurology, Tohoku University Graduate School of Medicine, Sendai, JapanMicro/Nano Technology Center, Tokai University, Hiratsuka, Kanagawa, JapanDepartment of Mechanical Engineering, Tokai University School of Engineering, Hiratsuka, Kanagawa, JapanMolecular Neuropathobiology Laboratory, Department of Physiology, Tokai University School of Medicine, Isehara, Kanagawa, JapanMicro/Nano Technology Center, Tokai University, Hiratsuka, Kanagawa, JapanIntroductionAmyotrophic 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.MethodsIn 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.Results and discussionsAfter 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.https://www.frontiersin.org/articles/10.3389/fncel.2025.1590732/fullamyotrophic lateral sclerosis (ALS)iPSCsmicrofluidic deviceFUS/TLSlower motor neurons |
| spellingShingle | Asako Otomo Asako Otomo Keiko Nishijima Yuta Murakami Mitsuru Ishikawa Mitsuru Ishikawa Haruka Yudahira Kento Shimakura Hideyuki Okano Hideyuki Okano Masashi Aoki Hiroshi Kimura Hiroshi Kimura Shinji Hadano Shinji Hadano Investigation of early axonal phenotypes in an iPSC-derived ALS cellular model using a microfluidic device Frontiers in Cellular Neuroscience amyotrophic lateral sclerosis (ALS) iPSCs microfluidic device FUS/TLS lower motor neurons |
| title | Investigation of early axonal phenotypes in an iPSC-derived ALS cellular model using a microfluidic device |
| title_full | Investigation of early axonal phenotypes in an iPSC-derived ALS cellular model using a microfluidic device |
| title_fullStr | Investigation of early axonal phenotypes in an iPSC-derived ALS cellular model using a microfluidic device |
| title_full_unstemmed | Investigation of early axonal phenotypes in an iPSC-derived ALS cellular model using a microfluidic device |
| title_short | Investigation of early axonal phenotypes in an iPSC-derived ALS cellular model using a microfluidic device |
| title_sort | investigation of early axonal phenotypes in an ipsc derived als cellular model using a microfluidic device |
| topic | amyotrophic lateral sclerosis (ALS) iPSCs microfluidic device FUS/TLS lower motor neurons |
| url | https://www.frontiersin.org/articles/10.3389/fncel.2025.1590732/full |
| work_keys_str_mv | AT asakootomo investigationofearlyaxonalphenotypesinanipscderivedalscellularmodelusingamicrofluidicdevice AT asakootomo investigationofearlyaxonalphenotypesinanipscderivedalscellularmodelusingamicrofluidicdevice AT keikonishijima investigationofearlyaxonalphenotypesinanipscderivedalscellularmodelusingamicrofluidicdevice AT yutamurakami investigationofearlyaxonalphenotypesinanipscderivedalscellularmodelusingamicrofluidicdevice AT mitsuruishikawa investigationofearlyaxonalphenotypesinanipscderivedalscellularmodelusingamicrofluidicdevice AT mitsuruishikawa investigationofearlyaxonalphenotypesinanipscderivedalscellularmodelusingamicrofluidicdevice AT harukayudahira investigationofearlyaxonalphenotypesinanipscderivedalscellularmodelusingamicrofluidicdevice AT kentoshimakura investigationofearlyaxonalphenotypesinanipscderivedalscellularmodelusingamicrofluidicdevice AT hideyukiokano investigationofearlyaxonalphenotypesinanipscderivedalscellularmodelusingamicrofluidicdevice AT hideyukiokano investigationofearlyaxonalphenotypesinanipscderivedalscellularmodelusingamicrofluidicdevice AT masashiaoki investigationofearlyaxonalphenotypesinanipscderivedalscellularmodelusingamicrofluidicdevice AT hiroshikimura investigationofearlyaxonalphenotypesinanipscderivedalscellularmodelusingamicrofluidicdevice AT hiroshikimura investigationofearlyaxonalphenotypesinanipscderivedalscellularmodelusingamicrofluidicdevice AT shinjihadano investigationofearlyaxonalphenotypesinanipscderivedalscellularmodelusingamicrofluidicdevice AT shinjihadano investigationofearlyaxonalphenotypesinanipscderivedalscellularmodelusingamicrofluidicdevice |