High Yield Derivation of Enriched Glutamatergic Neurons from Suspension-Cultured Mouse ESCs for Neurotoxicology Research
Abstract
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. 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, alpha-latrotoxin (LTX), and botulinum neurotoxin (BoNT), were also evaluated. Suspension-adapted ESCs expressed markers of pluripotency through at least 30 passages, and differentiation produced 97 10(6) 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.
Document Details
- Document Type
- Technical Report
- Publication Date
- Oct 24, 2012
- Accession Number
- ADA574506
Entities
People
- Ian M. Gut
- Kaylie M. Tuznik
- Kyle S. Hubbard
- Mariano T. Mesngon
- Megan E. Lyman
- Patrick M. McNutt
Organizations
- United States Army Medical Research Institute of Chemical Defense