Cross-Comparison of Human iPSC Motor Neuron Models of Familial and Sporadic ALS Reveals Early and Convergent Transcriptomic Disease Signatures.

Induced pluripotent stem cell (iPSC)-derived neural cultures from amyotrophic lateral sclerosis (ALS) patients can model disease phenotypes. However, heterogeneity arising from genetic and experimental variability limits their utility, impacting reproducibility and the ability to track cellular origins of pathogenesis. Here, we present methodologies using single-cell RNA sequencing (scRNA-seq) analysis to address these limitations. By repeatedly differentiating and applying scRNA-seq to motor neurons (MNs) from healthy, familial ALS, sporadic ALS, and genome-edited iPSC lines across multiple patients, batches, and platforms, we account for genetic and experimental variability toward identifying unified and reproducible ALS signatures. Combining HOX and developmental gene expression with global clustering, we anatomically classified cells into rostrocaudal, progenitor, and postmitotic identities. By relaxing statistical thresholds, we discovered genes in iPSC-MNs that were concordantly dysregulated in postmortem MNs and yielded predictive ALS markers in other human and mouse models. Our approach thus revealed early, convergent, and MN-resolved signatures of ALS.

Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro.

Human cell reprogramming technologies offer access to live human neurons from patients and provide a new alternative for modeling neurological disorders in vitro. Neural electrical activity is the essence of nervous system function in vivo. Therefore, we examined neuronal activity in media widely used to culture neurons. We found that classic basal media, as well as serum, impair action potential generation and synaptic communication. To overcome this problem, we designed a new neuronal medium (BrainPhys basal + serum-free supplements) in which we adjusted the concentrations of inorganic salts, neuroactive amino acids, and energetic substrates. We then tested that this medium adequately supports neuronal activity and survival of human neurons in culture. Long-term exposure to this physiological medium also improved the proportion of neurons that were synaptically active. The medium was designed to culture human neurons but also proved adequate for rodent neurons. The improvement in BrainPhys basal medium to support neurophysiological activity is an important step toward reducing the gap between brain physiological conditions in vivo and neuronal models in vitro.