POSTER link: BonnBrain 2019_Final_PR
Abstract:
HUMAN IPSC-DERIVED PNS MONO- & CO-CULTURE SYSTEMS FOR PAIN AND SENSORY NEURON RESEARCH AND DRUG DISCOVERY
In a physiological state pain is one of the most important senses in terms of survival and well being, but is very problematic when it develops under disease conditions into a chronic state. Sensation of pain is initiated by nociceptive peripheral sensory neurons. The cell bodies of sensory neurons cluster together with non-neuronal cells (glia, immune cells) in the dorsal root ganglion, from where signals are transmitted from the PNS to the CNS. Recently, non-neuronal cells emerged as key contributors to pathological and chronic pain mechanisms. Hence, biomedical research on pain mechanisms as well as drug screening activities would largely benefit from hiPSC-derived models that enable the robust and efficient derivation of pain relevant neuronal and glial cell types for i) in vitro modeling of neuron-glia interactions and ii) HTS compatible drug screening campaigns. In order to establish a HTS compatible platform for pain research and drug discovery we set up a scalable and robust differentiation protocol for the generation of cryopreservable hiPSC-derived sensory neurons (hiPSC-SN). The derived nociceptor cultures homogenously express nociceptive markers Brn3a (> 80 %), Isl1 (> 85 %) and TrkA (> 70 %) and were shown to exhibit normal resting membrane potential, induced action potential firing, lack of spontaneous action potentials as well as response to GABA, pH-shift and ATP. Culture conditions of hiPSC-SNs were adapted to HTS-formats to study the response of nociceptors to pain relevant stimuli on a FLIPR platform. We could show positive responses to agonists of TRPV1- and P2X3-receptors. To better model the in vivo situation we are aiming for more complex neuron-glia in vitro systems. Therefore we have implemented protocols for the derivation of iPSC-derived Schwann cells (hiPSC-SC) and myeloid cells (hiPSC-M) for the establishment of long-term hiPSC-SC/SN and acute hiPSC-M/SN co-cultures. Whilst hiPSC-SC/SN co-cultures are instrumental to examine the impact of cell-cell interaction on maturation and myelin-wrapping, hiPSC-M/SN co-cultures will provide a window to study the early phases of neuropathic pain development and the impact of released cytokines on pain sensitization.