Endocannabinoid signalling in stem cells and cerebral organoids drives differentiation to deep layer projection neurons via CB 1 receptors

The endocannabinoid (eCB) system, via cannabinoid CB₁ receptor, regulates neurodevelopment by controlling neural progenitor proliferation and neurogenesis. CB₁ receptor signalling in vivo drives corticofugal deep layer projection neuron development through the regulation of BCL11B and SATB2 transcription factors. Here, we investigated the role of eCB signalling in mouse pluripotent embryonic stem cell-derived neuronal differentiation. Characterization of the eCB system revealed increased expression of eCB-metabolizing enzymes, eCB ligands and CB₁ receptors along neuronal differentiation. CB₁ receptor knockdown inhibited neuronal differentiation of deep layer neurons and increased upper layer neuron generation, and this phenotype was rescued by CB₁ re-expression. Pharmacological regulation with CB₁ receptor agonists or elevation of eCB tone with a monoacylglycerol lipase inhibitor promoted neuronal differentiation of deep layer neurons at the expense of upper layer neurons. Patch-clamp analyses revealed that enhancing cannabinoid signalling facilitated neuronal differentiation and functionality. Noteworthy, incubation with CB₁ receptor agonists during human iPSC-derived cerebral organoid formation also promoted the expansion of BCL11B⁺ neurons. These findings unveil a cell-autonomous role of eCB signalling that, via CB₁ receptor, promotes mouse and human deep layer cortical neuron development.