FROM A PLACODE PROGENITOR PLATFORM TO A TRIGEMINAL GANGLION ORGANOID: MODELLING SENSORY DEVELOPMENT AND FUNCTION IN VITRO

The trigeminal ganglion (TG) is composed of peripheral sensory neurons which transmit pain, temperature and touch information from the face to the brain. Developmentally, it originates from an ectodermal population named pre-placodal ectoderm, which is marked by the expression of the transcription factor SIX1, and which gives rise to most of the facial peripheral nervous system. The intermediate region of the pre-placodal ectoderm later acquires a trigeminal fate, marked by the expression of PAX3, and thus develops into the TG. The TG is involved in several human pathologies, such as migraine and viral diseases, including the establishment of viral latency in trigeminal sensory neurons by herpesviruses. TG-associated pathologies remain poorly understood in part due to a lack of human, scalable in vitro models of the ganglion. Human pluripotent stem cells can be differentiated towards three-dimensional organoids mirroring tissue development, morphology and function, but organoids of the TG are yet to be developed. Here, we aim at generating the first TG organoid (TGO) and at characterising its development, maturation and function. By mimicking TG development in vitro, we successfully generated a multipotent placode progenitor organoid (PPO) composed almost exclusively of cells expressing the placodal marker SIX1. To guide PPOs towards an intermediate fate expressing PAX3, we performed a high-content chemical compound screen and identified novel signalling pathways important for trigeminal development. We thus obtained an organoid enriched in trigeminal progenitors, which, when cultured over time, developed into trigeminal sensory neurons. Characterisation of TGOs by scRNA-seq and spatial transcriptomics at defined developmental time points highlighted the emergence of distinct sensory neuron and glial subpopulations, which transcriptionally and spatially resembled in vivo TG cells. Importantly, the sensory neurons in TGOs are functional, as they are able to fire action potentials in response to pain and temperature-mimicking stimuli. TGOs thus closely resemble human TG in vivo in their development, cell composition, and function. They constitute the first human scalable system to investigate TG physiology and pathology, and could be employed in the future to shed light on diseases such as migraine and viral latency in the ganglion.