Swift Induction of Human Spinal Lower Motor Neurons and Robust ALS Cell Screening Via Single-cell Imaging

Overview

Journal Stem Cell Reports

Publisher Cell Press

Specialty Cell Biology

Date 2024 Dec 20

PMID 39706179

Authors

Selena Setsu

Satoru Morimoto

Shiho Nakamura

Fumiko Ozawa

Kagistia Hana Utami

Ayumi Nishiyama

Naoki Suzuki

Masashi Aoki

Yukio Takeshita

Yukihide Tomari

Hideyuki Okano

Affiliations

Soon will be listed here.

Abstract

This study introduces a novel method for rapidly and efficiently inducing human spinal lower motor neurons (LMNs) from induced pluripotent stem cells (iPSCs) to eventually elucidate the pathomechanisms of amyotrophic lateral sclerosis (ALS) and facilitate drug screening. Previous methods were limited by low induction efficiency, poor LMN purity, or labor-intensive induction and evaluation processes. Our protocol overcomes these challenges, achieving around 80% induction efficiency within just two weeks by combining a small molecule-based approach with transcription factor transduction. Moreover, to exclude non-LMN cells from the analysis, we utilized time-lapse microscopy and machine learning to analyze the morphology and viability of iPSC-derived LMNs on a single-cell basis, establishing an effective pathophysiological evaluation system. This rapid, efficient, and streamlined protocol, along with our single-cell-based evaluation method, enables large-scale analysis and drug screening using iPSC-derived motor neurons.

References

Park J, Kim J, Lewy T, Rice C, Elemento O, Rendeiro A . Spatial omics technologies at multimodal and single cell/subcellular level. Genome Biol. 2022; 23(1):256. PMC: 9746133. DOI: 10.1186/s13059-022-02824-6. View

Mattiazzi Usaj M, Yeung C, Friesen H, Boone C, Andrews B . Single-cell image analysis to explore cell-to-cell heterogeneity in isogenic populations. Cell Syst. 2021; 12(6):608-621. PMC: 9112900. DOI: 10.1016/j.cels.2021.05.010. View

Wetts R, Vaughn J . Choline acetyltransferase and NADPH diaphorase are co-expressed in rat spinal cord neurons. Neuroscience. 1994; 63(4):1117-24. DOI: 10.1016/0306-4522(94)90577-0. View

Bonaventura G, Iemmolo R, Attaguile G, La Cognata V, Pistone B, Raudino G . iPSCs: A Preclinical Drug Research Tool for Neurological Disorders. Int J Mol Sci. 2021; 22(9). PMC: 8123805. DOI: 10.3390/ijms22094596. View

Fujimori K, Ishikawa M, Otomo A, Atsuta N, Nakamura R, Akiyama T . Modeling sporadic ALS in iPSC-derived motor neurons identifies a potential therapeutic agent. Nat Med. 2018; 24(10):1579-1589. DOI: 10.1038/s41591-018-0140-5. View

Chen S, Zhou Y, Chen Y, Gu J . fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018; 34(17):i884-i890. PMC: 6129281. DOI: 10.1093/bioinformatics/bty560. View

Wainger B, Kiskinis E, Mellin C, Wiskow O, Han S, Sandoe J . Intrinsic membrane hyperexcitability of amyotrophic lateral sclerosis patient-derived motor neurons. Cell Rep. 2014; 7(1):1-11. PMC: 4023477. DOI: 10.1016/j.celrep.2014.03.019. View

Radivojevic M, Punga A . Functional imaging of conduction dynamics in cortical and spinal axons. Elife. 2023; 12. PMC: 10444024. DOI: 10.7554/eLife.86512. View

Xu H, Yao Y, Yao F, Chen J, Li M, Yang X . Generation of functional posterior spinal motor neurons from hPSCs-derived human spinal cord neural progenitor cells. Cell Regen. 2023; 12(1):15. PMC: 10033800. DOI: 10.1186/s13619-023-00159-6. View

10.

Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K . Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007; 131(5):861-72. DOI: 10.1016/j.cell.2007.11.019. View

11.

Palasantzas V, Tamargo-Rubio I, Le K, Slager J, Wijmenga C, Jonkers I . iPSC-derived organ-on-a-chip models for personalized human genetics and pharmacogenomics studies. Trends Genet. 2023; 39(4):268-284. DOI: 10.1016/j.tig.2023.01.002. View

12.

Ito D, Morimoto S, Takahashi S, Okada K, Nakahara J, Okano H . Maiden voyage: induced pluripotent stem cell-based drug screening for amyotrophic lateral sclerosis. Brain. 2022; 146(1):13-19. DOI: 10.1093/brain/awac306. View

13.

Takeda T, Iijima M, Shimizu Y, Yoshizawa H, Miyashiro M, Onizuka H . p.N345K mutation in TARDBP in a patient with familial amyotrophic lateral sclerosis: An autopsy case. Neuropathology. 2019; 39(4):286-293. DOI: 10.1111/neup.12559. View

14.

Okano H, Morimoto S . iPSC-based disease modeling and drug discovery in cardinal neurodegenerative disorders. Cell Stem Cell. 2022; 29(2):189-208. DOI: 10.1016/j.stem.2022.01.007. View

15.

Vance C, Rogelj B, Hortobagyi T, De Vos K, Nishimura A, Sreedharan J . Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science. 2009; 323(5918):1208-1211. PMC: 4516382. DOI: 10.1126/science.1165942. View

16.

Imamura K, Izumi Y, Watanabe A, Tsukita K, Woltjen K, Yamamoto T . The Src/c-Abl pathway is a potential therapeutic target in amyotrophic lateral sclerosis. Sci Transl Med. 2017; 9(391). DOI: 10.1126/scitranslmed.aaf3962. View

17.

Takahashi K, Yamanaka S . Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006; 126(4):663-76. DOI: 10.1016/j.cell.2006.07.024. View

18.

Santilli G, Lamorte G, Carlessi L, Ferrari D, Nodari L, Binda E . Mild hypoxia enhances proliferation and multipotency of human neural stem cells. PLoS One. 2010; 5(1):e8575. PMC: 2797394. DOI: 10.1371/journal.pone.0008575. View

19.

Schenke M, Prause H, Bergforth W, Przykopanski A, Rummel A, Klawonn F . Human-Relevant Sensitivity of iPSC-Derived Human Motor Neurons to BoNT/A1 and B1. Toxins (Basel). 2021; 13(8). PMC: 8402508. DOI: 10.3390/toxins13080585. View

20.

Arai T, Hasegawa M, Akiyama H, Ikeda K, Nonaka T, Mori H . TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem Biophys Res Commun. 2006; 351(3):602-11. DOI: 10.1016/j.bbrc.2006.10.093. View