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

Francesco Limone; Irune Guerra San Juan; 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

doi:10.1016/j.celrep.2022.111896

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.

Authors

Francesco Limone¹, Irune Guerra San Juan², Jana M Mitchell³, Janell L M Smith⁴, Kavya Raghunathan³, Daniel Meyer⁵, Sulagna Dia Ghosh⁶, Alexander Couto⁴, Joseph R Klim³, Brian J Joseph⁷, John Gold⁴, Curtis J Mello⁵, James Nemesh⁵, Brittany M Smith⁴, Matthijs Verhage⁸, Steven A McCarroll⁵, Olli Pietiläinen⁹, Ralda Nehme³, Kevin Eggan¹⁰

Affiliations

¹ Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Stanley Centre for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Leiden University Medical Center, LUMC, 2333 ZA Leiden, the Netherlands. Electronic address: [email protected].
² Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Stanley Centre for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Vrije Universiteit, Amsterdam, the Netherlands; Human Genetics, Amsterdam University Medical Center, Amsterdam, the Netherlands.
³ Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Stanley Centre for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
⁴ Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA.
⁵ Stanley Centre for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
⁶ Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Stanley Centre for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
⁷ Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Departments of Pathology and Cell Biology, Columbia University Irving Medical Centre, New York, NY 10032, USA.
⁸ Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Vrije Universiteit, Amsterdam, the Netherlands; Human Genetics, Amsterdam University Medical Center, Amsterdam, the Netherlands.
⁹ Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Stanley Centre for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Neuroscience Center, Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland.
¹⁰ Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA. Electronic address: [email protected].

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

Grants and funding

U01 MH115727/MH/NIMH NIH HHS/United States