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

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

^*Corresponding author for this work

Research output: Contribution to Journal › Article › Academic › peer-review

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.

Original language	English
Article number	111896
Pages (from-to)	1-21
Number of pages	21
Journal	Cell Reports
Volume	42
Issue number	1
Early online date	2 Jan 2023
DOIs	https://doi.org/10.1016/j.celrep.2022.111896
Publication status	Published - 31 Jan 2023

Bibliographical note

Funding Information:
We were supported by U01MH115727 to S.A.M., R.N., and K.E., as well as by the Stanley Center for Psychiatric Research at Broad Institute of MIT and Harvard, United States, and the Harvard Stem Cell and Regenerative Biology Department at Harvard University, United States .

Funding Information:
The authors thank members of the Eggan, McCarroll, Nehme, and Pietiläinen groups for insights and useful discussions and Lee Rubin and members of the Rubin laboratory for support in the final steps of this project. We are grateful to the Harvard Stem Cell and Regenerative Biology Department (HSCRB) for the use of shared equipment, microscopy core, and FACS facility. We are also grateful to the Stanley Center at the Broad Institute of Harvard and MIT for support with bioinformatics and cloud space. We thank BioRender.com for useful diagrams and images used in figures throughout this publication. We were supported by U01MH115727 to S.A.M. R.N. and K.E. as well as by the Stanley Center for Psychiatric Research at Broad Institute of MIT and Harvard, United States, and the Harvard Stem Cell and Regenerative Biology Department at Harvard University, United States. Conception and study design: F.L. and R.N. Data analysis and interpretation: F.L. O.P. and R.N. Manuscript writing and editing: F.L. O.P. R.N. and M.V. Village design and multi-cell line experiments: J.M. J.L.M.S. and K.R. Immunofluorescence analysis and co-culture studies: F.L. I.G.S.J. A.C. and B.M.S. Conventional motor neuron experiments: F.L. B.M.S. I.G.S.J. and J.R.K. Electrophysiology analysis: B.J.J. J.G. and F.L. Bioinformatics and scRNA-seq: F.L. S.M. O.P. S.D.G. D.M. C.J.M. and J.M. K.E. is cofounder of Q-State Biosciences, Quralis, Enclear Therapies, and is group vice president at BioMarin Pharmaceutical. We worked to ensure diversity in experimental samples through the selection of the cell lines. We worked to ensure diversity in experimental samples through the selection of the genomic datasets. One or more of the authors of this paper self-identifies as an under-represented ethnic minority in their field of research or within their geographical location. One or more of the authors of this paper self-identifies as a gender minority in their field of research. One or more of the authors of this paper self-identifies as a member of the LGBTQIA+ community.

Publisher Copyright:
© 2022 The Author(s)

Funding

We were supported by U01MH115727 to S.A.M., R.N., and K.E., as well as by the Stanley Center for Psychiatric Research at Broad Institute of MIT and Harvard, United States, and the Harvard Stem Cell and Regenerative Biology Department at Harvard University, United States . The authors thank members of the Eggan, McCarroll, Nehme, and Pietiläinen groups for insights and useful discussions and Lee Rubin and members of the Rubin laboratory for support in the final steps of this project. We are grateful to the Harvard Stem Cell and Regenerative Biology Department (HSCRB) for the use of shared equipment, microscopy core, and FACS facility. We are also grateful to the Stanley Center at the Broad Institute of Harvard and MIT for support with bioinformatics and cloud space. We thank BioRender.com for useful diagrams and images used in figures throughout this publication. We were supported by U01MH115727 to S.A.M. R.N. and K.E. as well as by the Stanley Center for Psychiatric Research at Broad Institute of MIT and Harvard, United States, and the Harvard Stem Cell and Regenerative Biology Department at Harvard University, United States. Conception and study design: F.L. and R.N. Data analysis and interpretation: F.L. O.P. and R.N. Manuscript writing and editing: F.L. O.P. R.N. and M.V. Village design and multi-cell line experiments: J.M. J.L.M.S. and K.R. Immunofluorescence analysis and co-culture studies: F.L. I.G.S.J. A.C. and B.M.S. Conventional motor neuron experiments: F.L. B.M.S. I.G.S.J. and J.R.K. Electrophysiology analysis: B.J.J. J.G. and F.L. Bioinformatics and scRNA-seq: F.L. S.M. O.P. S.D.G. D.M. C.J.M. and J.M. K.E. is cofounder of Q-State Biosciences, Quralis, Enclear Therapies, and is group vice president at BioMarin Pharmaceutical. We worked to ensure diversity in experimental samples through the selection of the cell lines. We worked to ensure diversity in experimental samples through the selection of the genomic datasets. One or more of the authors of this paper self-identifies as an under-represented ethnic minority in their field of research or within their geographical location. One or more of the authors of this paper self-identifies as a gender minority in their field of research. One or more of the authors of this paper self-identifies as a member of the LGBTQIA+ community.

Funders	Funder number
Broad Institute of Harvard
Harvard Stem Cell and Regenerative Biology Department
Harvard Stem Cell and Regenerative Biology Department at Harvard
University of California Institute for Mexico and the United States
Massachusetts Institute of Technology	U01MH115727
Massachusetts Institute of Technology
Harvard University
Stanley Center for Psychiatric Research, Broad Institute

Keywords

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

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10.1016/j.celrep.2022.111896

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Cite this

Limone, F., Guerra San Juan, I., Mitchell, J. M., Smith, J. L. M., Raghunathan, K., Meyer, D., Ghosh, S. D., Couto, A., Klim, J. R., Joseph, B. J., Gold, J., Mello, C. J., Nemesh, J., Smith, B. M., Verhage, M., McCarroll, S. A., Pietiläinen, O., Nehme, R., & Eggan, K. (2023). Efficient generation of lower induced motor neurons by coupling Ngn2 expression with developmental cues. Cell Reports, 42(1), 1-21. Article 111896. https://doi.org/10.1016/j.celrep.2022.111896

@article{b42ddbd62a5a4b06a091c237de0dd313,

title = "Efficient generation of lower induced motor neurons by coupling Ngn2 expression with developmental cues",

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, differentiation protocol, Dropulation, human stem cells, motor neuron, multiplexed pooled sequencing, neuronal differentiation, NGN2, patterning molecules, single cell profiling, spinal cord",

author = "Francesco Limone and \{Guerra San Juan\}, Irune and Mitchell, \{Jana M.\} and Smith, \{Janell L.M.\} and Kavya Raghunathan and Daniel Meyer and Ghosh, \{Sulagna Dia\} and Alexander Couto and Klim, \{Joseph R.\} and Joseph, \{Brian J.\} and John Gold and Mello, \{Curtis J.\} and James Nemesh and Smith, \{Brittany M.\} and Matthijs Verhage and McCarroll, \{Steven A.\} and Olli Pietil{\"a}inen and Ralda Nehme and Kevin Eggan",

note = "Funding Information: We were supported by U01MH115727 to S.A.M., R.N., and K.E., as well as by the Stanley Center for Psychiatric Research at Broad Institute of MIT and Harvard, United States, and the Harvard Stem Cell and Regenerative Biology Department at Harvard University, United States . Funding Information: The authors thank members of the Eggan, McCarroll, Nehme, and Pietil{\"a}inen groups for insights and useful discussions and Lee Rubin and members of the Rubin laboratory for support in the final steps of this project. We are grateful to the Harvard Stem Cell and Regenerative Biology Department (HSCRB) for the use of shared equipment, microscopy core, and FACS facility. We are also grateful to the Stanley Center at the Broad Institute of Harvard and MIT for support with bioinformatics and cloud space. We thank BioRender.com for useful diagrams and images used in figures throughout this publication. We were supported by U01MH115727 to S.A.M. R.N. and K.E. as well as by the Stanley Center for Psychiatric Research at Broad Institute of MIT and Harvard, United States, and the Harvard Stem Cell and Regenerative Biology Department at Harvard University, United States. Conception and study design: F.L. and R.N. Data analysis and interpretation: F.L. O.P. and R.N. Manuscript writing and editing: F.L. O.P. R.N. and M.V. Village design and multi-cell line experiments: J.M. J.L.M.S. and K.R. Immunofluorescence analysis and co-culture studies: F.L. I.G.S.J. A.C. and B.M.S. Conventional motor neuron experiments: F.L. B.M.S. I.G.S.J. and J.R.K. Electrophysiology analysis: B.J.J. J.G. and F.L. Bioinformatics and scRNA-seq: F.L. S.M. O.P. S.D.G. D.M. C.J.M. and J.M. K.E. is cofounder of Q-State Biosciences, Quralis, Enclear Therapies, and is group vice president at BioMarin Pharmaceutical. We worked to ensure diversity in experimental samples through the selection of the cell lines. We worked to ensure diversity in experimental samples through the selection of the genomic datasets. One or more of the authors of this paper self-identifies as an under-represented ethnic minority in their field of research or within their geographical location. One or more of the authors of this paper self-identifies as a gender minority in their field of research. One or more of the authors of this paper self-identifies as a member of the LGBTQIA+ community. Publisher Copyright: {\textcopyright} 2022 The Author(s)",

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doi = "10.1016/j.celrep.2022.111896",

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Limone, F, Guerra San Juan, I, Mitchell, JM, Smith, JLM, Raghunathan, K, Meyer, D, Ghosh, SD, Couto, A, Klim, JR, Joseph, BJ, Gold, J, Mello, CJ, Nemesh, J, Smith, BM, Verhage, M, McCarroll, SA, Pietiläinen, O, Nehme, R & Eggan, K 2023, 'Efficient generation of lower induced motor neurons by coupling Ngn2 expression with developmental cues', Cell Reports, vol. 42, no. 1, 111896, pp. 1-21. https://doi.org/10.1016/j.celrep.2022.111896

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T1 - Efficient generation of lower induced motor neurons by coupling Ngn2 expression with developmental cues

AU - Limone, Francesco

AU - Guerra San Juan, Irune

AU - Mitchell, Jana M.

AU - Smith, Janell L.M.

AU - Raghunathan, Kavya

AU - Meyer, Daniel

AU - Ghosh, Sulagna Dia

AU - Couto, Alexander

AU - Klim, Joseph R.

AU - Joseph, Brian J.

AU - Gold, John

AU - Mello, Curtis J.

AU - Nemesh, James

AU - Smith, Brittany M.

AU - Verhage, Matthijs

AU - McCarroll, Steven A.

AU - Pietiläinen, Olli

AU - Nehme, Ralda

AU - Eggan, Kevin

N1 - Funding Information: We were supported by U01MH115727 to S.A.M., R.N., and K.E., as well as by the Stanley Center for Psychiatric Research at Broad Institute of MIT and Harvard, United States, and the Harvard Stem Cell and Regenerative Biology Department at Harvard University, United States . Funding Information: The authors thank members of the Eggan, McCarroll, Nehme, and Pietiläinen groups for insights and useful discussions and Lee Rubin and members of the Rubin laboratory for support in the final steps of this project. We are grateful to the Harvard Stem Cell and Regenerative Biology Department (HSCRB) for the use of shared equipment, microscopy core, and FACS facility. We are also grateful to the Stanley Center at the Broad Institute of Harvard and MIT for support with bioinformatics and cloud space. We thank BioRender.com for useful diagrams and images used in figures throughout this publication. We were supported by U01MH115727 to S.A.M. R.N. and K.E. as well as by the Stanley Center for Psychiatric Research at Broad Institute of MIT and Harvard, United States, and the Harvard Stem Cell and Regenerative Biology Department at Harvard University, United States. Conception and study design: F.L. and R.N. Data analysis and interpretation: F.L. O.P. and R.N. Manuscript writing and editing: F.L. O.P. R.N. and M.V. Village design and multi-cell line experiments: J.M. J.L.M.S. and K.R. Immunofluorescence analysis and co-culture studies: F.L. I.G.S.J. A.C. and B.M.S. Conventional motor neuron experiments: F.L. B.M.S. I.G.S.J. and J.R.K. Electrophysiology analysis: B.J.J. J.G. and F.L. Bioinformatics and scRNA-seq: F.L. S.M. O.P. S.D.G. D.M. C.J.M. and J.M. K.E. is cofounder of Q-State Biosciences, Quralis, Enclear Therapies, and is group vice president at BioMarin Pharmaceutical. We worked to ensure diversity in experimental samples through the selection of the cell lines. We worked to ensure diversity in experimental samples through the selection of the genomic datasets. One or more of the authors of this paper self-identifies as an under-represented ethnic minority in their field of research or within their geographical location. One or more of the authors of this paper self-identifies as a gender minority in their field of research. One or more of the authors of this paper self-identifies as a member of the LGBTQIA+ community. Publisher Copyright: © 2022 The Author(s)

PY - 2023/1/31

Y1 - 2023/1/31

N2 - 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.

AB - 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.

KW - CP: Neuroscience

KW - CP: Stem cell research

KW - differentiation protocol

KW - Dropulation

KW - human stem cells

KW - motor neuron

KW - multiplexed pooled sequencing

KW - neuronal differentiation

KW - NGN2

KW - patterning molecules

KW - single cell profiling

KW - spinal cord

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DO - 10.1016/j.celrep.2022.111896

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AN - SCOPUS:85147211184

SN - 2211-1247

VL - 42

SP - 1

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JO - Cell Reports

JF - Cell Reports

IS - 1

M1 - 111896

ER -

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

Abstract

Bibliographical note

Funding

Keywords

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