Efficient generation of lower induced Motor Neurons by coupling Ngn2 expression with developmental cues. 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. Overall design: 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/