Author:
Hubbard Kyle S,Gut Ian M,Lyman Megan E,Tuznik Kaylie M,Mesngon Mariano T,McNutt Patrick M
Abstract
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.
Publisher
Springer Science and Business Media LLC
Subject
Cellular and Molecular Neuroscience,General Neuroscience
Reference45 articles.
1. Eubanks LM, Hixon MS, Jin W, Hong S, Clancy CM, Tepp WH, Baldwin MR, Malizio CJ, Goodnough MC, Barbieri JT, et al: An in vitro and in vivo disconnect uncovered through high-throughput identification of botulinum neurotoxin A antagonists. Proc Natl Acad Sci U S A. 2007, 104 (8): 2602-2607. 10.1073/pnas.0611213104.
2. Purkiss JR, Friis LM, Doward S, Quinn CP: Clostridium botulinum neurotoxins act with a wide range of potencies on SH-SY5Y human neuroblastoma cells. Neurotoxicology. 2001, 22 (4): 447-453. 10.1016/S0161-813X(01)00042-0.
3. Durham HD, Dahrouge S, Cashman NR: Evaluation of the spinal cord neuron X neuroblastoma hybrid cell line NSC-34 as a model for neurotoxicity testing. Neurotoxicology. 1993, 14 (4): 387-395.
4. Clementi E, Raichman M, Meldolesi J: Heterogeneity of NGF-induced differentiation in PC12 cells investigated in a battery of isolated cell clones. Funct Neurol. 1993, 8 (2): 109-113.
5. NIAID: Summary of the NIAID expert panel on botulinum neurotoxins therapeutics. NIAID expert panel on botulinum neurotoxins therapeutics. 2004, National Institute of Allergy and Infectious Diseases, Bethesda, MD
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