Human iPSC-derived neurons: a surrogate for native human DRG neurons
Human induced pluripotent stem cell-derived neurons (human iPSC-derived neurons) allow to develop and study CNS models in a relevant genetic and cellular context. But to what extent can human iPSC-derived sensory neuron be used as surrogate for native human DRG neurons to support a preclinical drug discovery program in Pain? What are the electrophysiological properties or these human iPSC-derived sensory neurons?
To answer these questions, Neuroservice USA developed and optimized a robust assay of the electrical excitability of human iPSC-derived neurons, by following these steps:
1. Develop and validate an in vitro electrophysiology assay to measure the electrical excitability of human iPSC-derived sensory neurons.
2. Verify the functional expression of Kv7 using retigabine to open K channels to:
a. Hyperpolarize the resting membrane potential.
b. Decrease the number of evoked spikes elicited by depolarizing current injections.
3. Verify the functional expression of Kv7 using XE-991 to reverse the effect of retigabine.
MATERIAL & METHODS
Human iPSC-derived sensory neuron progenitors (Axol Bioscience) were thawed and plated at low density on a confluent monolayer of rat astrocytes on glass coverslips. The media was supplemented with MAXIMIZER, GDNF, NGF, BDNF, and NT-3 to promote the maturation of the cells to a differentiated sensory neuron phenotype. Recordings were made on cells ≥20 days in vitro.
Patch clamp electrophysiology
Standard patch clamp methods were employed. The external recording solution composition 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 internal recording solution composition was: 120 mM K-gluconate, 20 mM KCl, 3 mM MgCl2, 5 mM EGTA, 0.5 mM CaCl2, 10 mM HEPES pH 7.3
Retigabine was prepared as a 10 mM DMSO stock solution and diluted 1:1000 in external recording solution to make a 10 uM working solution.
XE-991 (Sigma) was prepared as a 10 mM DMSO stock solution and diluted 1:1000 in external recording solution to make a 10 uM working solution.
Retigabine hyperpolarized the resting membrane potential and decreased action potential firing in human iPSC-derived sensory neurons .
We have developed and optimized a robust assay of the electrical excitability of human iPSC-derived sensory neurons.
This assay can be used to examine the structure activity relationships (SAR) of a series of Kv7 channel openers.