Efficient generation of lower induced Motor Neurons by coupling Ngn2 expression with developmental cues

Human pluripotent stem cells (hPSCs) are a powerful tool for disease modelling 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 factor Neurogenin2 (Ngn2) with small molecule patterning to differentiate hPSCs into lower induced Motor Neurons (liMoNes/liMNs). This approach induces canonical MN markers including motor neuron (MN) specific marker Hb9/MNX1 activation in >95% of cells. liMNs resemble bona fide hPSC-derived MN, exhibit spontaneous electrical activity, express synaptic markers and can contact muscle cells in vitro. Pooled, multiplexed single-cell RNA sequencing on 50 hPSC-lines reveals reproducible populations of distinct subtypes of cervical 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 disease. Pooled "villages" of lower induced Motor Neurons differentited from human stem-cells (n=50, two timepoints= day 35 and day 49 post-induction) were analyzed using scRNAseq Submitter states: Raw data are available at: https://duos.broadinstitute.org/