Neurotrophic requirements of human motor neurons defined using amplified and purified stem cell-derived cultures

PLoS One. 2014 Oct 22;9(10):e110324. doi: 10.1371/journal.pone.0110324. eCollection 2014.

Abstract

Human motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC(50) 1-2 pM). The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening.

Publication types

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

MeSH terms

  • 1-Methyl-3-isobutylxanthine / pharmacology
  • Amides / pharmacology
  • Brain-Derived Neurotrophic Factor / pharmacology*
  • Cell Differentiation / drug effects
  • Cell Proliferation / drug effects
  • Cell Survival / drug effects
  • Cells, Cultured
  • Ciliary Neurotrophic Factor / pharmacology*
  • Colforsin / pharmacology
  • Cyclic AMP / agonists
  • Cyclic AMP / metabolism
  • Embryonic Stem Cells / cytology
  • Embryonic Stem Cells / drug effects*
  • Embryonic Stem Cells / enzymology
  • Gene Expression
  • Glial Cell Line-Derived Neurotrophic Factor / pharmacology*
  • Humans
  • Induced Pluripotent Stem Cells / cytology
  • Induced Pluripotent Stem Cells / drug effects*
  • Induced Pluripotent Stem Cells / enzymology
  • Motor Neurons / cytology
  • Motor Neurons / drug effects*
  • Motor Neurons / enzymology
  • Protein Kinase Inhibitors / pharmacology
  • Pyridines / pharmacology
  • rho-Associated Kinases / antagonists & inhibitors
  • rho-Associated Kinases / genetics
  • rho-Associated Kinases / metabolism

Substances

  • Amides
  • Brain-Derived Neurotrophic Factor
  • Ciliary Neurotrophic Factor
  • Glial Cell Line-Derived Neurotrophic Factor
  • Protein Kinase Inhibitors
  • Pyridines
  • Y 27632
  • Colforsin
  • BDNF protein, human
  • Cyclic AMP
  • rho-Associated Kinases
  • 1-Methyl-3-isobutylxanthine

Grants and funding

This work was funded by Project A.L.S., P2ALS and NYSTEM grant number CO24415. The work of N.J.L. was supported by the Portuguese Foundation for Science and Technology SFRH/BD/33421/2008 and the Luso-American Development Foundation. B.J.-K. was supported by the National Institute of Neurological Disorders and Stroke (NINDS). L.R. was supported by the Swedish Brain Foundation/Hjärnfonden. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.