Human Induced Pluripotent Stem Cell-Derived 3D-Neurospheres are Suitable for Neurotoxicity Screening

Cells. 2020 May 1;9(5):1122. doi: 10.3390/cells9051122.


We present a hiPSC-based 3D in vitro system suitable to test neurotoxicity (NT). Human iPSCs-derived 3D neurospheres grown in 96-well plate format were characterized timewise for 6-weeks. Changes in complexity and homogeneity were followed by immunocytochemistry and transmission electron microscopy. Transcriptional activity of major developmental, structural, and cell-type-specific markers was investigated at weekly intervals to present the differentiation of neurons, astrocytes, and oligodendrocytes. Neurospheres were exposed to different well-known toxicants with or without neurotoxic effect (e.g., paraquat, acrylamide, or ibuprofen) and examined at various stages of the differentiation with an ATP-based cell viability assay optimized for 3D-tissues. Concentration responses were investigated after acute (72 h) exposure. Moreover, the compound-specific effect of rotenone was investigated by a panel of ER-stress assay, TUNEL assay, immunocytochemistry, electron microscopy, and in 3D-spheroid based neurite outgrowth assay. The acute exposure to different classes of toxicants revealed distinct susceptibility profiles in a differentiation stage-dependent manner, indicating that hiPSC-based 3D in vitro neurosphere models could be used effectively to evaluate NT, and can be developed further to detect developmental neurotoxicity (DNT) and thus replace or complement the use of animal models in various basic research and pharmaceutical applications.

Keywords: 3D culture; induced pluripotent stem cells; neurite outgrowth; neurospheres; neurotoxicity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers / metabolism
  • Cell Death / drug effects
  • Cell Differentiation / drug effects
  • Cell Line
  • Cell Proliferation / drug effects
  • Cell Survival / drug effects
  • Female
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Induced Pluripotent Stem Cells / drug effects
  • Induced Pluripotent Stem Cells / ultrastructure
  • Middle Aged
  • Mitochondria / drug effects
  • Mitochondria / metabolism
  • Mitochondria / ultrastructure
  • Models, Biological
  • Neural Stem Cells / cytology
  • Neural Stem Cells / drug effects
  • Neuronal Outgrowth / drug effects
  • Neurons / cytology*
  • Neurons / drug effects
  • Neurons / ultrastructure
  • Neurotoxicity Syndromes / diagnosis*
  • Rotenone / toxicity
  • Spheroids, Cellular / cytology*
  • Spheroids, Cellular / drug effects
  • Spheroids, Cellular / ultrastructure


  • Biomarkers
  • Rotenone