Human iPSC-derived in vitro models of the peripheral nervous system for cellular pain and somatosensation research

Abstract

The term 'somatosensation' summarizes the sensation of touch, body position, temperature and pain experienced by animals or humans. Peripheral sensory neurons can be classified according to their primary sensation as nociceptors (pain), mechanoreceptors (pressure) or proprioceptors (body position). The cell bodies of sensory neurons cluster together with non-neuronal cells like satellite glia, mast cells, resident macrophages and Schwann cells in the dorsal root ganglion (DRG). Recent research revealed relevant differences between rodent and human sensory neuron function and subtype specification, highlighting the importance of human-specific models for pain and somatosensation research. This thesis aimed at the establishment of in vitro systems resembling different aspects of the peripheral nervous system and to explore the individual roles of neurons and glia. First, a scalable and robust differentiation protocol, based on a published small molecule approach, for the generation of cryopreservable iPSC-derived sensory neurons (smSNs) was implemented. SmSNs express the pan-sensory neural markers peripherin, BNR3A and ISLET1, as well as the nociceptive neural markers TRKA and TRPV1. SmSNs were employed in an image-based high-content screening assay, monitoring intracellular PKA-II signaling, as well as chronic pain disease modeling. To model the multicellular composition within the DRG, defined co-cultures of smSNs with iPSC-derived Schwann cells and macrophages were established, focusing on the functional changes of sensory neurons upon co-culture. To achieve a more complex representation of the human DRG in vitro, preliminary experiments were performed aiming at the generation of a three-dimensional, self-organized spheroid model resembling the multicellular DRG composition. Second a forward programming protocol was established, employing transcription factors relevant for the in vivo development of peripheral sensory neurons. Overexpression of NGN1 and BRN3A (NB) or NGN1, BRN3A and ISLET1 (NBI) in iPSC yields sensory neurons after only seven days. Both combinations yielded sensory neurons expressing key markers such as PRPH, TRPV1 and Nav1.7. Both forward programmed sensory neuron populations were able to recapitulate features of a genetic pain disorder, based on a multi-electrode array assay. The model systems implemented and characterized in this study enable diverse biomedical applications. Sensory neuron mono-cultures are assay and high-through put ready and can be employed to study cell autonomous defects such as sodium channel mutations and intracellular signaling processes. Defined co-cultures and DRG-like spheroids will be important tools for the identification and investigation of non-cell autonomous effects.