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

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.

Graphical abstract

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

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

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

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.