Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation

Abstract

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform highthroughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/ eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.

Low-Density Neuronal Cultures from Human Induced Pluripotent Stem Cells

“…5). Consistent with previous work using Matrigel-coated coverslips [14], most cells in low-density regions responded to AMPA receptor and GABA receptor stimulation (online suppl. Fig.…”

“…We utilized a strategy that enables large-scale generation of NSCs/NPCs from iPSCs [14]. NSCs were distributed at clonal density on both uncoated and Matrigel-coated coverslips as described in Materials and Methods (Fig.…”

“…Monolayer cultures of NSCs/NPCs were generated from iPSCs as previously described [14]. NSCs/NPCs were dissociated with Accutase, resuspended in mTeSR™1 medium, and seeded at the same density on uncoated or Matrigel-coated glass coverslips (4 × 10 5 cells/well in 12-well plates).…”

Impact of regulatory variation across human iPSCs and differentiated cells

“…These properties make iPSCs a valuable cellular model for the study of gene regulation in a controlled setting. Although some debate remains about whether iPSCs are truly equivalent to embryonic stem cells (ESCs), studies have shown, using wellmatched lines, that iPSCs are nearly indistinguishable from ESCs in their molecular profiles and their ability to differentiate (D'Aiuto et al 2014;Pagliuca et al 2014;Choi et al 2015;Davidson et al 2015).…”

Induced Pluripotent Stem Cell-Derived Human Glutamatergic Neurons as a Platform for Mechanistic Assessment of Inducible Excitotoxicity in Drug Discovery

“…The most important tasks in using iPSC-derived human glutamatergic neurons is achieving largescale generation of human iPSC-derived neural stem cells/early neural progenitor cells in spite of several challenges related to the differentiation procedures [7]. Terminal differentiation coupled with preliminary cell model qualification steps often reveal how different iPSC lines made from a single donor exhibit similar phenotypic marker expression as well as sensitivity to selected neurotoxicants.…”