Human induced pluripotent stem cells (hiPSCs) are expected to be a strong tool in drug discovery process. We have been attempting the establishment of the evaluation system of neuron-specific toxicity, i.e., excitotoxicity. In this study, we examined the conformities of two lines of hiPSC-derived neurons, i.e. neurons differentiated from 253G1 via EBs (embryoid bodies) and neurospheres, and iCell® neurons (iNeurons) commercially-available hiPSC-derived neurons. We quantified the cytotoxicity by three kinds of assays, i.e. propidium iodide (PI)/calcein staining, LDH assay, and MTT assay, which we have optimized for hiPSC-derived neurons.
253G1-derived neurons showed Ca²⁺ responses to L-Glu from 10 day in vitro (DIV), but did not respond to NMDA until 40 DIV. Staurosporine (10 μM, 48 hrs), an apoptosis inducer, increased the number of PI positive cells, LDH leakage, and decreased the number of calcein positive cells, MTT reduction at 7, 14, and 21 DIV. However, L-Glu (100 μM, 1 hr) did not cause neurotoxicity. We obtained same results in three independent experiments.
iNeurons showed Ca²⁺ responses to L-Glu from 1 DIV, however, the responsiveness to AP5, NMDA receptor blocker, was varied. Staurosporine reproducibly caused significant neurotoxicity, however, the neurotoxicity caused by L-Glu was also varied as well as AP5-responsiveness. The reproducibility of the results was low.
In our study so far, we have established the protocols for detecting neuronal damage, however, search for hiPSC-derived neurons, which stably express NMDA receptors, is necessary to establish the evaluation system of excitotoxicity.