Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Sep 19;6(3):104016.
doi: 10.1016/j.xpro.2025.104016. Epub 2025 Aug 6.

Protocol for the induction of human spinal motor neurons from human induced pluripotent stem cells for studying amyotrophic lateral sclerosis

Selena Setsu  1 , Satoru Morimoto  2 , Shiho Nakamura  2 , Fumiko Ozawa  2 , Hideyuki Okano  3
Affiliations

Protocol for the induction of human spinal motor neurons from human induced pluripotent stem cells for studying amyotrophic lateral sclerosis

Selena Setsu et al. STAR Protoc. .

Abstract

Here, we present a protocol for inducing spinal lower motor neurons (LMNs) from human induced pluripotent stem cells (iPSCs). We describe steps for preparation of a chemically induced transitional state (CTraS), transduction with Sendai virus, and LMN differentiation and maintenance. We then detail procedures for live imaging for single-cell-based survival analysis and neurite length of LMNs using BioStation and immunocytochemistry for induction efficiency check. This protocol is optimized for amyotrophic lateral sclerosis (ALS) research and large-scale screening. For complete details on the use and execution of this protocol, please refer to Setsu et al.1.

Keywords: Antibody; Cell Biology; Cell Differentiation; Cell culture; Cell-based Assays; Gene Expression; Microscopy; Molecular Biology; Neuroscience; Single Cell; Stem Cells.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests H.O. reports grants and personal fees from K Pharma, Inc., and SanBio Co., Ltd., outside the submitted work.

Figures

None
Graphical abstract
Figure 1
Figure 1
Progression of CTraS induction As induction progresses, each colony expands, and the center of the colony becomes elevated. Scale bar: 100 μm.
Figure 2
Figure 2
Progression of MN induction Cell morphology changes markedly during the first week. By day 7, cells exhibit typical neuronal characteristics, including extended neurites. Scale bar: 100 μm. An example of SeV vector construction is illustrated.
Figure 3
Figure 3
GFP expression 3 and 7 days after SeV transduction Scale bar: 160 μm. FP: fluorescent protein. Figures adopted from Setsu et al. with permission from Elsevier.
Figure 4
Figure 4
Functional, morphological, and survival phenotypes of induced lower motor neurons derived from iPSCs (A) Left: Representative immunocytochemistry image of induced lower motor neurons (LMNs) at day 7, stained for HB9 (magenta), ISLET1 (magenta), and TUBB3 (green), with Hoechst nuclear counterstaining (cyan). Scale bar = 10 μm. Right: Differentiation efficiency at day 7, quantified based on HB9 and ISLET1 immunostaining. Error bars represent standard deviation; n = 8 biological replicates (different iPSC lines). (B) Gene expression levels normalized by z-score; n = 3 technical replicates. (C) Total spike count, reflecting local field potential activity, measured over a 5-min MEA recording. Data represent mean ± SEM from 3 independent wells. (D) Weighted mean firing rate, calculated as the average firing rate adjusted for the number of active electrodes. Data represent mean ± SEM from 3 independent wells. (E) Total neurite length per well, normalized to the corresponding neurite length at day 3. (F) Survival analysis comparing ALS and wild-type control cell lines. P-values were determined using the log-rank test with Benjamini–Hochberg correction. All panels are reproduced or adopted from Setsu et al. with permission from Elsevier.
See this image and copyright information in PMC

References

    1. Setsu S., Morimoto S., Nakamura S., Ozawa F., Utami K.H., Nishiyama A., Suzuki N., Aoki M., Takeshita Y., Tomari Y., Okano H. Swift induction of human spinal lower motor neurons and robust ALS cell screening via single-cell imaging. Stem Cell Rep. 2025;20 doi: 10.1016/j.stemcr.2024.11.007. - DOI - PMC - PubMed
    1. Fujimori K., Matsumoto T., Kisa F., Hattori N., Okano H., Akamatsu W. Escape from Pluripotency via Inhibition of TGF-β/BMP and Activation of Wnt Signaling Accelerates Differentiation and Aging in hPSC Progeny Cells. Stem Cell Rep. 2017;9:1675–1691. doi: 10.1016/j.stemcr.2017.09.024. - DOI - PMC - PubMed
    1. Takahashi K., Tanabe K., Ohnuki M., Narita M., Ichisaka T., Tomoda K., Yamanaka S. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell. 2007;131:861–872. doi: 10.1016/j.cell.2007.11.019. - DOI - PubMed
    1. Egawa N., Kitaoka S., Tsukita K., Naitoh M., Takahashi K., Yamamoto T., Adachi F., Kondo T., Okita K., Asaka I., et al. Drug Screening for ALS Using Patient-Specific Induced Pluripotent Stem Cells. Sci. Transl. Med. 2012;4 doi: 10.1126/scitranslmed.3004052. - DOI - PubMed
    1. Santilli G., Lamorte G., Carlessi L., Ferrari D., Rota Nodari L., Binda E., Delia D., Vescovi A.L., De Filippis L. Mild Hypoxia Enhances Proliferation and Multipotency of Human Neural Stem Cells. PLoS One. 2010;5 doi: 10.1371/journal.pone.0008575. - DOI - PMC - PubMed

MeSH terms