title: High yield derivation of enriched glutamatergic neurons from suspension-cultured mouse ESCs for neurotoxicology research.
abstract: BACKGROUND
  Recently, there has been a strong emphasis on identifying an in vitro model for neurotoxicity research that combines the biological relevance of primary neurons with the scalability, reproducibility and genetic tractability of continuous cell lines. Derived neurons should be homotypic, exhibit neuron-specific gene expression and morphology, form functioning synapses and consistently respond to neurotoxins in a fashion indistinguishable from primary neurons. However, efficient methods to produce neuronal populations that are suitable alternatives to primary neurons have not been available.
  
  
  METHODS
  With the objective of developing a more facile, robust and efficient method to generate enriched glutamatergic neuronal cultures, we evaluated the neurogenic capacity of three mouse embryonic stem cell (ESC) lines (R1, C57BL/6 and D3) adapted to feeder-independent suspension culture. Neurogenesis and neuronal maturation were characterized as a function of time in culture using immunological, genomic, morphological and functional metrics. The functional responses of ESNs to neurotropic toxins with distinctly different targets and mechanisms of toxicity, such as glutamate, α-latrotoxin (LTX), and botulinum neurotoxin (BoNT), were also evaluated.
  
  
  RESULTS
  Suspension-adapted ESCs expressed markers of pluripotency through at least 30 passages, and differentiation produced 97×106 neural progenitor cells (NPCs) per 10-cm dish. Greater than 99% of embryonic stem cell-derived neurons (ESNs) expressed neuron-specific markers by 96 h after plating and rapidly developed complex axodendritic arbors and appropriate compartmentalization of neurotypic proteins. Expression profiling demonstrated the presence of transcripts necessary for neuronal function and confirmed that ESN populations were predominantly glutamatergic. Furthermore, ESNs were functionally receptive to all toxins with sensitivities and responses consistent with primary neurons.
  
  
  CONCLUSIONS
  These findings demonstrate a cost-effective, scalable and flexible method to produce a highly enriched glutamatergic neuron population. The functional characterization of pathophysiological responses to neurotropic toxins and the compatibility with multi-well plating formats were used to demonstrate the suitability of ESNs as a discovery platform for molecular mechanisms of action, moderate-throughput analytical approaches and diagnostic screening. Furthermore, for the first time we demonstrate a cell-based model that is sensitive to all seven BoNT serotypes with EC50 values comparable to those reported in primary neuron populations. These data providing compelling evidence that ESNs offer a neuromimetic platform suitable for the evaluation of molecular mechanisms of neurotoxicity.
authors.affiliation: United States Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Rd, Aberdeen Proving Ground, MD 21010, USA. patrick.mcnutt@us.army.mil
authors.forename: Kyle S
authors.initials: KS
authors.lastname: Hubbard
authors.forename: Ian M
authors.initials: IM
authors.lastname: Gut
authors.forename: Megan E
authors.initials: ME
authors.lastname: Lyman
authors.forename: Kaylie M
authors.initials: KM
authors.lastname: Tuznik
authors.forename: Mariano T
authors.initials: MT
authors.lastname: Mesngon
authors.forename: Patrick M
authors.initials: PM
authors.lastname: McNutt
mesh_terms: D000818:Animals; D019274:Botulinum Toxins, Type A / Q000633:toxicity; D002118:Calcium / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D018698:Glutamic Acid / Q000378:metabolism* / Q000633:toxicity; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism; D055495:Neurogenesis / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D009498:Neurotoxins / Q000633:toxicity; D013111:Spider Venoms / Q000633:toxicity; D014116:Toxicology / Q000379:methods*
chemical_list: D009419:Nerve Tissue Proteins; D009498:Neurotoxins; D013111:Spider Venoms; D018698:Glutamic Acid; C020763:alpha-latrotoxin; D019274:Botulinum Toxins, Type A; D002118:Calcium