Human iPSC-derived neurons promise to be a relevant in vitro model of human neuronal function and are hypothesized to predict the efficacy of novel therapeutics for neuroscience indications. The phenotypes of hiPSC-derived neurons have been primarily characterized by mRNA expression. Very few reports describe the functional properties by electrophysiological methods. We describe the functional properties of an NGN2-derived cell line by both patch clamp and microelectrode array recording (HD-MEA).
NGN2 neurons were plated at low density on a confluent monolayer of rat astrocytes. The acquisition of a mature electrophysiological phenotype was monitored with time in culture. The neurons exhibited a negative resting membrane potential very early. Not surprisingly, the neurons grew larger with time in culture as measured by increased whole cell capacitance and decreased input resistance. The percentage of neurons exhibiting spontaneous action potential firing also increased with time in culture. Spontaneous postsynaptic currents were also detected, indicating that the NGN2 neurons formed functional synapses. We detected NMDA receptor currents in ~50% of the NGN2 neurons.
We also profiled the activity of NGN2 neurons using the MaxTwo HD-MEA (MaxWell Biosciences) that simultaneously records up to 1,020 neurons from 6 independent wells. In agreement with the patch clamp results, spontaneous activity increased with time in culture. The mean firing rate was ~1 Hz. NGN2 neurons in culture exhibited network connectivity as seen by coherent bursting with a frequency of ~0.2 Hz and a burst duration of ~0.8 sec. Co-culturing the excitatory NGN2 neurons with human iCellGABA inhibitory neurons (Fujifilm) decreased the spontaneous activity of NGN2 neurons.
METHODS
- Human NGN2 neurons and E18 rat cortical neurons were plated at a very low density (50-100 cells/mm2) on monolayers of rat astrocytes.
- Whole-cell patch clamp recordings were made using standard methods. The composition of the external recording solution was: 140 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1.3 mM MgCl2, 10 mM glucose, 10 mM HEPES pH 7.3. The composition of the internal recording solution was: 120 mM K-gluconate, 20 mM KCl, 3 mM MgCl2, 5 mM EGTA, 0.5 mM CaCl2, 4 mM Na2-ATP, 0.3 mM Li-GTP, 10 mM HEPES pH 7.3.
- We measured spontaneous action potentials using the Maxwell MaxTwo high-density multielectrode array (HD-MEA). The development of spontaneous action potential firing was monitored from 1-7 weeks in vitro using the Activity Scan recording protocol (40 sec recording). Bursting activity was recorded from 1,020 electrodes simultaneously per well using the Network recording protocol (5 min recording).
HUMAN IPSC-DERIVED NGN2 NEURONS RESEMBLE THE MORPHOLOGY OF PRIMARY RAT CNS NEURONS
FUNCTIONAL PROPERTIES OF HUMAN IPSC-DERIVED NGN2 NEURONS AND PRIMARY RAT CNS NEURONS
SPONTANEOUS ACTION POTENTIALS AND NETWORK BURSTING IN PRIMARY RAT CNS NEURONS AND HUMAN NGN2 NEURONS
COMPARISON OF THE NETWORK BURSTING ENDPOINTS IN HUMAN NGN2 NEURONS AND RAT CNS NEURONS
NETWORK BURSTING ENDPOINTS IN HUMAN NGN2 NEURONS AND RAT CNS NEURONS
SUMMARY
1.Human iPSC-derived NGN2 neurons are suitable for patch clamp recording and MEA recording endpoints.
2.Human NGN2 neurons exhibited functional properties expected of “bona fide” neurons isolated from rat CNS.
3.Human NGN2 neurons responded to test compounds (adenosine and CX614) as expected from primary rat neuron cultures.
4.Human NGN2 neurons can be used to support lead optimization (SAR) for drug discovery programs.
ADENOSINE REDUCES ACTIVITY AND CX614 INCREASES ACTIVITY IN HUMAN NGN2 NEURONS
EFFECT OF GABAZINE IN NGN2-ICELL GABA CO-CULTURES
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