High yield derivation of enriched glutamatergic neurons from suspension-cultured mouse ESCs for neurotoxicology research

Abstract

BackgroundRecently, 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.MethodsWith 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.ResultsSuspension-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.ConclusionsThese 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.

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

“…By DIV 14 synapsin-1 + puncta can be identified at axodendritic interfaces, suggestive of synapse formation. This is consistent with the expression of synaptic marker proteins prior to DIV 7 8,11 . Neuronal morphologies continue to mature through DIV 21, at which time cultures exhibit elaborate axodendritic arbors and large synaptic puncta (Figure 2A).…”

“…Longitudinal expression profiling using RNA-sequencing corroborated morphological and proteomic evidence of neuronal specification and maturation 11,15 . Representative markers of developmental progression exhibited stage-specific expression profiles, including Oct3/4, Nestin, DCX, NeuN and KCC2 ( Figure 2B).…”

“…We have developed a differentiation protocol that enables the economic production of large quantities of highly pure stem cell-derived neurons from mouse ESCs (detailed in Figure 1A) 8,11 . This method has been used over a period of years to reproducibly differentiate privately generated and commercially available ESC lines into neurons of defined lineages [12][13][14] .…”

“…MIST was used to measure the effects of BoNT/A-/G or TeNT on synaptic activity in ESNs at 20 hr after bath addition of each toxin. Toxins were added at a concentration equivalent to 10-fold the EC 50 value, as previously determined by immunoblot analysis of SNARE protein cleavage 11 . All toxins reduced mEPSC frequencies to less than 5% of vehicle-treated controls.…”

“…In particular, mouse stem cellderived neurons (ESNs) have been found to be highly sensitive to physiological doses of BoNT/A-/G and to replicate many in vivo responses to intoxication, including differential persistences, activity-enhanced intoxication, and serotype-specific potencies 5,8,11 . These behaviors suggest that in vivo mechanisms of BoNT uptake, processing, trafficking and activity are conserved in ESNs.…”