Efficient generation of lower induced motor neurons by coupling Ngn2 expression with developmental cues

• DOI: 10.1016/j.celrep.2022.111896
• Publication Date: 2023-01-02T15:47:15Z
• Authors: Francesco Limone; Irune Guerra San ...; Jana M. Mitchell; Janell L.M. Smith; Kavya Raghunathan; Daniel Meyer; Sulagna Dia Ghosh; Alexander Couto; Joseph R. Klim; Brian J. Joseph; John Gold; Curtis J. Mello; James Nemesh; Brittany M. Smith; Matthijs Verhage; Steven A. McCarroll; Olli Pietiläinen; Ralda Nehme; Kevin Eggan
• Keywords: multiplexed pooled sequencing; Direct conversion; Induced Pluripotent Stem Cells; Pluripotent stem-cells; Dropulation; Article; NGN2; CP: Stem cell research; Identity; Humans; motor neuron; neuronal differentiation; Biochemistry, cell and molecular biology; differentiation protocol; Motor Neurons; Homeodomain Proteins; patterning molecules; spinal cord; Cell Differentiation; human stem cells; Motoneurons; Gene Expression Regulation; Spinal-cord; Differentiation; CP: Neuroscience; Functional-neurons; single cell profiling; Als; Cues; Inductive signals; Specification; Transcription Factors

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 factor 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 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.