Image 3_Investigation of early axonal phenotypes in an iPSC-derived ALS cellular model using a microfluidic device.tif
Introduction<p>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...
Shranjeno v:
| Glavni avtor: | |
|---|---|
| Drugi avtorji: | , , , , , , , , |
| Izdano: |
2025
|
| Teme: | |
| Oznake: |
Označite
Brez oznak, prvi označite!
|
| _version_ | 1851484332008407040 |
|---|---|
| author | Asako Otomo (251531) |
| author2 | Keiko Nishijima (21776255) Yuta Murakami (2178398) Mitsuru Ishikawa (606461) Haruka Yudahira (21776258) Kento Shimakura (21776261) Hideyuki Okano (47690) Masashi Aoki (35088) Hiroshi Kimura (3849) Shinji Hadano (251529) |
| author2_role | author author author author author author author author author |
| author_facet | Asako Otomo (251531) Keiko Nishijima (21776255) Yuta Murakami (2178398) Mitsuru Ishikawa (606461) Haruka Yudahira (21776258) Kento Shimakura (21776261) Hideyuki Okano (47690) Masashi Aoki (35088) Hiroshi Kimura (3849) Shinji Hadano (251529) |
| author_role | author |
| dc.creator.none.fl_str_mv | Asako Otomo (251531) Keiko Nishijima (21776255) Yuta Murakami (2178398) Mitsuru Ishikawa (606461) Haruka Yudahira (21776258) Kento Shimakura (21776261) Hideyuki Okano (47690) Masashi Aoki (35088) Hiroshi Kimura (3849) Shinji Hadano (251529) |
| dc.date.none.fl_str_mv | 2025-07-24T05:33:57Z |
| dc.identifier.none.fl_str_mv | 10.3389/fncel.2025.1590732.s007 |
| dc.relation.none.fl_str_mv | https://figshare.com/articles/figure/Image_3_Investigation_of_early_axonal_phenotypes_in_an_iPSC-derived_ALS_cellular_model_using_a_microfluidic_device_tif/29633705 |
| dc.rights.none.fl_str_mv | CC BY 4.0 info:eu-repo/semantics/openAccess |
| dc.subject.none.fl_str_mv | Cellular Interactions (incl. Adhesion, Matrix, Cell Wall) amyotrophic lateral sclerosis (ALS) iPSCs microfluidic device FUS/TLS lower motor neurons |
| dc.title.none.fl_str_mv | Image 3_Investigation of early axonal phenotypes in an iPSC-derived ALS cellular model using a microfluidic device.tif |
| dc.type.none.fl_str_mv | Image Figure info:eu-repo/semantics/publishedVersion image |
| description | Introduction<p>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.</p>Methods<p>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.</p>Results and discussions<p>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.</p> |
| eu_rights_str_mv | openAccess |
| id | Manara_24dceaf3a9b6dea7b3f7fc7b6e34e028 |
| identifier_str_mv | 10.3389/fncel.2025.1590732.s007 |
| network_acronym_str | Manara |
| network_name_str | ManaraRepo |
| oai_identifier_str | oai:figshare.com:article/29633705 |
| publishDate | 2025 |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | CC BY 4.0 |
| spelling | Image 3_Investigation of early axonal phenotypes in an iPSC-derived ALS cellular model using a microfluidic device.tifAsako Otomo (251531)Keiko Nishijima (21776255)Yuta Murakami (2178398)Mitsuru Ishikawa (606461)Haruka Yudahira (21776258)Kento Shimakura (21776261)Hideyuki Okano (47690)Masashi Aoki (35088)Hiroshi Kimura (3849)Shinji Hadano (251529)Cellular Interactions (incl. Adhesion, Matrix, Cell Wall)amyotrophic lateral sclerosis (ALS)iPSCsmicrofluidic deviceFUS/TLSlower motor neuronsIntroduction<p>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.</p>Methods<p>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.</p>Results and discussions<p>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.</p>2025-07-24T05:33:57ZImageFigureinfo:eu-repo/semantics/publishedVersionimage10.3389/fncel.2025.1590732.s007https://figshare.com/articles/figure/Image_3_Investigation_of_early_axonal_phenotypes_in_an_iPSC-derived_ALS_cellular_model_using_a_microfluidic_device_tif/29633705CC BY 4.0info:eu-repo/semantics/openAccessoai:figshare.com:article/296337052025-07-24T05:33:57Z |
| spellingShingle | Image 3_Investigation of early axonal phenotypes in an iPSC-derived ALS cellular model using a microfluidic device.tif Asako Otomo (251531) Cellular Interactions (incl. Adhesion, Matrix, Cell Wall) amyotrophic lateral sclerosis (ALS) iPSCs microfluidic device FUS/TLS lower motor neurons |
| status_str | publishedVersion |
| title | Image 3_Investigation of early axonal phenotypes in an iPSC-derived ALS cellular model using a microfluidic device.tif |
| title_full | Image 3_Investigation of early axonal phenotypes in an iPSC-derived ALS cellular model using a microfluidic device.tif |
| title_fullStr | Image 3_Investigation of early axonal phenotypes in an iPSC-derived ALS cellular model using a microfluidic device.tif |
| title_full_unstemmed | Image 3_Investigation of early axonal phenotypes in an iPSC-derived ALS cellular model using a microfluidic device.tif |
| title_short | Image 3_Investigation of early axonal phenotypes in an iPSC-derived ALS cellular model using a microfluidic device.tif |
| title_sort | Image 3_Investigation of early axonal phenotypes in an iPSC-derived ALS cellular model using a microfluidic device.tif |
| topic | Cellular Interactions (incl. Adhesion, Matrix, Cell Wall) amyotrophic lateral sclerosis (ALS) iPSCs microfluidic device FUS/TLS lower motor neurons |