Rationale:
Ion channel studies have long used recombinant human proteins expressed in human cell lines to screen ion channel modulators and to develop potential disease-modifying drug candidates. Although recombinant human proteins are powerful tools, they have several limitations such as missing co-expression of appropriate accessory subunits, lack of full excitable cell context and the difficulty to examine complex endogenous channel stoichiometry and gene expression. Human induced pluripotent stem cells (hiPSCs) encapsulate the signaling pathways of the desired cell types, such as neurons, and thus they are better model cells for defining the critical ion channel activity in healthy and disease states. As such, hiPSC married with high-throughput automated patch clamp (APC) ion channel recording platforms provide a foundation for significant physiological, medical and drug discovery advances.
Methods:
hiPSC NGN2 neurons were provided by Biogen. Standard operating procedures for cell culturing and cell suspension preparation were based on Sophion standards or provided by Biogen. Solutions: Extracellular solution (in mM): 2 CaCl2, 1 MgCl2, 10 HEPES, 4 KCl, 145 NaCl, 10 Glucose, pH = 7.4 with KOH, osmolarity 305 mOsm with sucrose. Potassium-based intracellular solution (in mM): 120 KF, 20 KCl, 10 HEPES, 10 EGTA, pH 7.2 with KOH, osmolarity 300 mOsm with sucrose. Cesium based intracellular solution (in mM): 140 CsF, 1EGTA, 5 CsOH, 10 HEPES, 10 NaCl, pH: 7.3 with CsOH, osmolarity 320 mOsm with sucrose. Voltage- and current-clamp protocols: Holding potential was at -90mV, voltage step protocol consisted of a 200 ms pre-step at -120 mV, followed by 500 ms voltage steps from -90 mV to +60 mV with 10mV increment. In step current clamp, holding voltage was at -90mV, followed by a sequential 10 pA stepwise current injection. All recordings were performed on the Qube 384 system of Sophion Bioscience. All analysis was performed with Sophion Analyzer.
Results:
Electrophysiological recordings following optimized dissociation found that ~30% of single cells had NaV currents >200 pA, leading to recordings of >100 cells in parallel. Minimal reduction of experimental throughput was observed with recordings following culture up to four weeks. Isolation of NaV currents with cesium internal solution showed expected NaV activation and inactivation curves with mean NaV currents >1 nA. Exchange of intracellular solution from cesium to potassium-based reversed block of Kv channels and did not significantly impact recording success rate. In multi-cell recording configurations we attained success rates of ~80%, sufficient to examine dozens of experimental conditions simultaneously.
Conclusions:
Our data demonstrate the feasibility of performing cell type and ion channel characterization with the combination of hiPSC-derived NGN2 neurons and automated patch clamp platforms which is comparable with manual patch clamp recordings. These results reveal the fundamental hiPSC neuronal properties obtained from automated patch clamp system, with drastically increased throughput relative to manual recordings.
Funding: This work was supported by Sophion Bioscience