Derivation of Spinal Cord Organoids as a quantitative approach to study the mammalian Hedgehog Signalling v2

The Hedgehog (HH) pathway is crucial for embryonic development, and adult homeostasis. Its dysregulation is implicated in multiple diseases. Existing cellular models used to study HH signal regulation in mammals do not fully recapitulate the complexity of the pathway. Here we describe the procedure to derive Spinal Cord Organoids (SCOs) and we show how to apply them to quantitively study the activity of the HH pathway. During SCO formation, the specification of different categories of neural progenitors (NPC) depends on the intensity of the HH signal, mirroring the process that occurs during neural tube development. By assessing the number of NPCs within these distinct subgroups, we are able to categorize and quantify the activation level of the HH pathway. SCOs represent an accessible and reliablein-vitrotool to quantify HH signaling and investigate the contribution of genetic and chemical cues in the HH pathway regulation.