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Human pluripotent stem cells (hPSCs) are a powerful tool for disease modeling of hard-to-access tissues (such as the brain). Current protocols either direct neuronal differentiation with small molecules or use transcription-factor-mediated programming. In this study, we couple overexpression of transcription fac-tor Neurogenin2 (Ngn2) with small molecule patterning to differentiate hPSCs into lower induced motor neurons (liMoNes/liMNs). This approach induces canonical MN markers including MN-specific Hb9/ MNX1 in more than 95% of cells. liMNs resemble bona fide hPSC-derived MN, exhibit spontaneous elec-trical activity, express synaptic markers, and can contact muscle cells in vitro. Pooled, multiplexed sin-gle-cell RNA sequencing on 50 hPSC lines reveals reproducible populations of distinct subtypes of cer-vical and brachial MNs that resemble their in vivo, embryonic counterparts. Combining small molecule patterning with Ngn2 overexpression facilitates high-yield, reproducible production of disease-relevant MN subtypes, which is fundamental in propelling our knowledge of MN biology and its disruption in dis-ease.

SUMMARY Dysregulated axonal trafficking of mitochondria is linked to neurodegenerative disorders. We report a high-content screen for small-molecule regulators of the axonal transport of mitochondria. Six compounds enhanced mitochondrial transport in the sub-micromolar range, acting via three cellular targets: F-actin, Tripeptidyl peptidase 1 (TPP1), or Aurora Kinase B (AurKB). Pharmacological inhibition or small hairpin RNA (shRNA) knockdown of each target promotes mitochondrial axonal transport in rat hippocampal neurons and induced pluripotent stem cell (iPSC)-derived human cortical neurons and enhances mitochondrial transport in iPSC-derived motor neurons from an amyotrophic lateral sclerosis (ALS) patient bearing one copy of SOD1A4V mutation. Our work identifies druggable regulators of axonal transport of mitochondria, provides broadly applicable methods for similar image-based screens, and suggests that restoration of proper axonal trafficking of mitochondria can be achieved in human ALS neurons., Graphical Abstract, In Brief Shlevkov et al. establish a high-content screen for enhancers of axonal mitochondrial trafficking. Identified compounds act through three cellular targets: F-Actin, Tripeptidyl peptidase 1, and Aurora Kinase B. Motor neurons derived from a SOD1+/A4VALS patient have decreased mitochondrial motility, which can be reversed by inhibitors of these targets.