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

The endocannabinoid (eCB) system, via the cannabinoid CB receptor, regulates neurodevelopment by controlling neural progenitor proliferation and neurogenesis. CB receptor signalling 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 CB receptors during neuronal differentiation. CB receptor knockdown inhibited neuronal differentiation of deep layer neurons and increased upper layer neuron generation, and this phenotype was rescued by CB re-expression. Pharmacological regulation with CB 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 CB 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 the CB receptor, promotes mouse and human deep layer cortical neuron development.