Category: Assay Development and Screening
Physiologically relevant human models of chronic pain are essential to developing new therapeutics and overcome poor translation between animal studies and the clinical setting. We therefore developed a humanized screening assay with sensory neurons derived from human induced pluripotent stem cells (hiPSCs). The resulting assay can be applied in screening to identify compounds that change a disease phenotype, such as neuronal excitability, rather than the activity of specific targets.
Human iPSCs were obtained from the European bank of induced stem cells (EBiSC) and a working bank of hiPSCs was produced. We differentiated the hiPSCs by dual SMAD inhibition followed by further neural commitment and patterning. Neural crest cells were cryopreserved and quality control performed to determine quality and yield of neuronal cultures. Neural crest cells were subsequently matured into functioning neurons using growth factors to induce sensory neuronal development.
To determine the optimal cell density and time in culture for the assay, sensory neuronal progenitors were plated at four different densities and electric field stimulation (EFS) experiments were run after 2, 3 and 5 weeks in culture. On the day of the experiment the cells were incubated with a calcium probe and an electrode array was used to electrically excite the neurons and simultaneously an integrated imaging-based microplate reader was used to monitor the calcium response of the neurons.
At the gene and protein level expression, markers for of neural crest, peripheral and sensory neurons were used (e.g. SOX10, BRN3A, NEUROG1, OTX2)as well as markers for pain-relevant neuronal targets such as SCN9A, SCN10A, NTRK1, and TRPV1.
Using optimal settings identified during assay development, the EFS assay performance was stable and the protocols used selectively and specifically stimulated voltage-gated sodium channels. To determine whether the assay could be used for single concentration screening, it was validated by testing a LOPAC library (Sigma-Aldrich) in neurons from different batches. There was a good agreement between the replicates demonstrating assay robustness and capability to support primary screening.
In conclusion, we have identified an in vitro, human assay approach utilizing human iPSC-derived sensory neurons and a physiological stimulus that yields highly reproducible and sensitive responses in a high capacity format. This enables true phenotypic screening of compound libraries using a highly relevant stimulus (electric field) in human sensory neurons.
Johan Pihl– VP Technology and Engineering, Cellectricon, Molndal, Vastra Gotaland, Sweden
VP Technology and Engineering
Molndal, Vastra Gotaland, Sweden
Johan joined Cellectricon in 2005, and has been in R&D and commercial positons at the company. He has been involved in the development of Cellectricon’s microfluidics-based electrophysiology platform for recombinant cell lines and primary neuronal cells, and managed the development of Cellectricon’s electromanipulation platform, Cellaxess Elektra. He currently holds the position of VP of Technology & Engineering at Cellectricon, and is responsible for developing Cellectricons internal technology platform for Discovery Services. Johan has a PhD in Chemistry from Chalmers University of Technology, and is the author and co-author of a number of scientific publications and patents.