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

Cell Rep. 2023 Jan 31;42(1):111896. doi: 10.1016/j.celrep.2022.111896. Epub 2023 Jan 2.

Abstract

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.

Keywords: CP: Neuroscience; CP: Stem cell research; Dropulation; NGN2; differentiation protocol; human stem cells; motor neuron; multiplexed pooled sequencing; neuronal differentiation; patterning molecules; single cell profiling; spinal cord.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Differentiation
  • Cues*
  • Gene Expression Regulation
  • Homeodomain Proteins / metabolism
  • Humans
  • Induced Pluripotent Stem Cells* / metabolism
  • Motor Neurons / metabolism
  • Transcription Factors / metabolism

Substances

  • Transcription Factors
  • MNX1 protein, human
  • Homeodomain Proteins