Combining NGN2 Programming with Developmental Patterning Generates Human Excitatory Neurons with NMDAR-Mediated Synaptic Transmission
Ralda Nehme,
Emanuela Zuccaro,
Sulagna Dia Ghosh,
Chenchen Li,
John L. Sherwood,
Olli Pietilainen,
Lindy E. Barrett,
Francesco Limone,
Kathleen A. Worringer,
Sravya Kommineni,
Ying Zang,
Davide Cacchiarelli,
Alex Meissner,
Rolf Adolfsson,
Stephen Haggarty,
Jon Madison,
Matthias Muller,
Paola Arlotta,
Zhanyan Fu,
Guoping Feng,
Kevin Eggan
Affiliations
Ralda Nehme
Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA
Emanuela Zuccaro
Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA
Sulagna Dia Ghosh
Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA
Chenchen Li
Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
John L. Sherwood
Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA
Olli Pietilainen
Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA
Lindy E. Barrett
Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA
Francesco Limone
Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA
Kathleen A. Worringer
Novartis Institutes for Biomedical Research, Novartis, Cambridge, MA 02139, USA
Sravya Kommineni
Novartis Institutes for Biomedical Research, Novartis, Cambridge, MA 02139, USA
Ying Zang
Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
Davide Cacchiarelli
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA
Alex Meissner
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA
Rolf Adolfsson
Umea University, Faculty of Medicine, Department of Clinical Sciences, Psychiatry, 901 85 Umea, Sweden
Stephen Haggarty
Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA
Jon Madison
Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
Matthias Muller
Novartis Institutes for Biomedical Research, Novartis, 4056 Basel, Switzerland
Paola Arlotta
Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA
Zhanyan Fu
Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
Guoping Feng
Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; McGovern Institute for Brain Research in the Department of Brain and Cognitive Sciences at MIT, Cambridge, MA 02139, USA
Kevin Eggan
Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Corresponding author
Summary: Transcription factor programming of pluripotent stem cells (PSCs) has emerged as an approach to generate human neurons for disease modeling. However, programming schemes produce a variety of cell types, and those neurons that are made often retain an immature phenotype, which limits their utility in modeling neuronal processes, including synaptic transmission. We report that combining NGN2 programming with SMAD and WNT inhibition generates human patterned induced neurons (hpiNs). Single-cell analyses showed that hpiN cultures contained cells along a developmental continuum, ranging from poorly differentiated neuronal progenitors to well-differentiated, excitatory glutamatergic neurons. The most differentiated neurons could be identified using a CAMK2A::GFP reporter gene and exhibited greater functionality, including NMDAR-mediated synaptic transmission. We conclude that utilizing single-cell and reporter gene approaches for selecting successfully programmed cells for study will greatly enhance the utility of hpiNs and other programmed neuronal populations in the modeling of nervous system disorders. : Nehme et al. combine two strong neuralizing factors (transcription factor programming and small molecule patterning) to generate human excitatory neurons from stem cells. They further undertake single-cell and reporter gene approaches to select highly differentiated neurons with increased functionality, augmenting their utility in the modeling of nervous system disorders. Keywords: human stem cell, neuronal differentiation, NGN2, dual SMAD inhibition, Wnt inhibition, excitatory neurons, single cell profiling, AMPAR, NMDAR, CAMK2A
