Human sensorimotor organoids derived from healthy and amyotrophic lateral sclerosis stem cells form neuromuscular junctions

Human induced pluripotent stem cells (iPSC) hold promise for modeling diseases in individual human genetic backgrounds and thus for developing precision medicine. Here, we generate sensorimotor organoids containing physiologically functional neuromuscular junctions (NMJs) and apply the model to different subgroups of amyotrophic lateral sclerosis (ALS). Using a range of molecular, genomic, and physiological techniques, we identify and characterize motor neurons and skeletal muscle, along with sensory neurons, astrocytes, microglia, and vasculature. Organoid cultures derived from multiple human iPSC lines generated from individuals with ALS and isogenic lines edited to harbor familial ALS mutations show impairment at the level of the NMJ, as detected by both contraction and immunocytochemical measurements. The physiological resolution of the human NMJ synapse, combined with the generation of major cellular cohorts exerting autonomous and non-cell autonomous effects in motor and sensory diseases, may prove valuable to understand the pathophysiological mechanisms of ALS.


Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used CRISPR-Cas9 edited iPSC lines carrying the ALS associated mutations TDP+/G298S, PFN1+/G118V, SOD1+/G85R, and paired isotype controls are available upon request.
Sample sizes were not pre-determined. We selected sample sizes according to existing examples in the field.
Raw sequence reads were processed using the bcbio-nextgen single-cell RNA-seq pipeline (https://bcbio-nextgen.readthedocs.io/en/latest/ contents/pipelines.html#single-cell-rna-seq). The pipeline uses tools from the umis repository (https://github.com/vals/umis) to generate a cell by gene count matrix. FASTQ files were formatted to parse out non-biological segments of the reads (i.e. cellular barcode, sample barcodes, and UMIs). Excess cellular barcodes were removed to reduce artifacts. The reads were aligned to the GRCh38 Ensembl Release 90 transcriptome with RapMap 84. Duplicate UMIs were collapsed and the number of reads per transcript were counted for each cellular barcode. Samples were assessed for quality and filtered using the distributions of reads per cell, UMIs per cell, genes per cell, mitochondrial ratios per cell, UMIs vs. genes detected, UMIs vs. read counts, and novelty scores. Cell clustering was performed using the Seurat R package (v 2.3.4)23,85 in R(3.5). Cells with less than 200 unique genes were removed from the analysis, and genes expressed in less than five cells were filtered out. Raw expression values were log normalized and each gene was scaled and centered after regression of contributions from batch, cell cycle phase, total number of reads, and number of mitochondrial genes. SPRING analysis was performed using the webtool35: number of PCA dimensions chosen was 50, with gene filtering settings of a minimum count of 3, a variability percentile threshold of 80%, and 5 nearest neighbors. No minimum was applied for cell filtering and a minimum of 3 cells was used for gene filtering.
These are standard filtering criteria for single-cell RNA-seq, as is the exclusion of cells with less than 200 unique genes.
All experiments were ran at independent times (n reported per experiment) and results such as contractions were confirmed across all lines in independent differentiations. Experiments were conducted in a minimum of 3 biological replicas (independent differentiations).
Randomization was not used because experiments generally did not involve treatment assignment. In the small number of experiments involving treatment of wells from an individual iPSC line with drugs, assignment was done in a blind, arbitrary manner.
Experimental acquisition and analysis were all performed with full blinding.