Combining NGN2 Programming With Developmental Patterning Generates Human Excitatory Neurons With NMDAR-Mediated Synaptic Transmission

Cell Rep. 2018 May 22;23(8):2509-2523. doi: 10.1016/j.celrep.2018.04.066.

Abstract

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.

Keywords: AMPAR; CAMK2A; NGN2; NMDAR; Wnt inhibition; dual SMAD inhibition; excitatory neurons; human stem cell; neuronal differentiation; single cell profiling.

Publication types

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

MeSH terms

  • Adult
  • Basic Helix-Loop-Helix Transcription Factors / metabolism*
  • Body Patterning*
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2 / metabolism
  • Cell Differentiation
  • Cells, Cultured
  • Fetus / cytology
  • Gene Expression Regulation
  • Humans
  • Nerve Tissue Proteins / metabolism*
  • Neurons / cytology
  • Neurons / metabolism*
  • Pluripotent Stem Cells / cytology
  • Pluripotent Stem Cells / metabolism
  • Receptors, AMPA / metabolism
  • Receptors, Glutamate / metabolism
  • Receptors, N-Methyl-D-Aspartate / metabolism*
  • Smad Proteins / metabolism
  • Synapses / metabolism
  • Synaptic Transmission*
  • Time Factors
  • Transcription, Genetic
  • Wnt Proteins / metabolism

Substances

  • Basic Helix-Loop-Helix Transcription Factors
  • NEUROG2 protein, human
  • Nerve Tissue Proteins
  • Receptors, AMPA
  • Receptors, Glutamate
  • Receptors, N-Methyl-D-Aspartate
  • Smad Proteins
  • Wnt Proteins
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2