Patch-clamp electrophysiology assays are routinely used to characterize neuronal excitability and provide functional endpoints to assess the effects of drug compounds. However, the basic electrophysiological properties of human iPSC-derived sensory neurons have not been well studied.
How well do iPSC-derived sensory neurons reflect the physiological state of native primary sensory neurons? How consistent is the phenotype? How reproducible are the data?
To address these questions, we focused on human sensory neurons differentiated from iPSC-derived neural progenitors (Axol Bioscience, ax0055). We conducted a thorough patch-clamp analysis, including: 1) current-clamp recordings to measure spontaneous and evoked action potential firing and 2) voltage-clamp recordings to characterize tetrodotoxin-sensitive and tetrodotoxin-resistant Na currents (found in native neurons).
We also determined the concentration-response of tetracaine, a voltage- and frequency-dependent blocker of voltage gated Na currents. We recorded >500 neurons from twelve independent neural precursor differentiation experiments.
We conclude that human iPSC-derived sensory neurons reproduce several key electrophysiological properties expected from bona fide dorsal root ganglia sensory neurons and are useful for supporting drug discovery programs for pain.
Cell culture
Frozen vials of human iPSC-derived sensory neuron progenitors (Axol Bioscience ax0055) were stored in the vapor phase of liquid nitrogen.
Plating Media, Sensory Neuron Maintenance Media, neurotropic factors, and Maximizer supplement were also provided by Axol as part of the complete cell culture kit. Sensory neuron progenitors were thawed and plated at high density (113,000 cells/cm2) on poly-D-lysine (PDL) coated glass coverslips in Neural Plating Medium (Axol). Alternatively, cells were plated at low density (15,000 cells/cm2) on a confluent monolayer of rat cortical astrocytes growing on glass cover slips. The next day, the Neural Plating Medium was replaced with Sensory Neuron Maintenance Medium (Axol) supplemented with pen/strep, Maximizer, NGF (25 ng/ml), GDNF (25 ng/ml), BDNF (10 ng/ml), and NT-3 (10 ng/ml). On day 3, the cells were treated with mitomycin C (2.5 ug/ml) for 2 hours to arrest the growth and proliferation of dividing cells that did not differentiate into non-dividing neurons. After 2 hours, the medium was completely changed to remove the mitomycin C. On day 5, the medium was completely changed. Thereafter, the medium was refreshed by a ½ medium change three times per week.
Electrophysiology
Coverslips containing sensory neurons were transferred to an inverted microscope (Olympus IX73). Recordings were made using a Multiclamp 700B patch clamp amplifier (Axon) and signals digitized using a Digidata 1550B (Axon). PClamp 11 software was used for data acquisition (Clampex) and analysis (Clampfit). Cells were perfused by gravity with an external recording solution at room temperature at a flow rate of 1.5 ml/min. The external recording solution was comprised of (mM): 140 NaCl, 2.5 KCl, 2 CaCl2, 1.3 MgCl2, 10 glucose, 10 HEPES buffered to pH 7.3.
Standard patch clamp methods were used for whole-cell voltage clamp recordings. For voltage clamp recordings of Na currents, a Cs-based internal recording solution was used, comprised of (mM): 70 CsCl, 70 CsF, 3 MgCl2, 5 EGTA, 0.5 CaCl2, 10 HEPES buffered to pH 7.3. For current clamp recordings of action potentials, patch pipettes were filled with a K-based internal recording solution comprised of (mM): 120 K-gluconate, 20 KCl, 3 MgCl2, 5 EGTA, 0.5 CaCl2, 10 HEPES buffered to pH 7.3.
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