Endocannabinoid signalling in stem cells and cerebral organoids drives differentiation to deep layer projection neurons via CB1 receptors

<p>The endocannabinoid (eCB) system, <i>via</i> cannabinoid CB₁ receptor, regulates neurodevelopment by controlling neural progenitor proliferation and neurogenesis. CB₁ receptor signalling <i>in vivo</i> drives corticofugal deep layer projection neuron development through the regulation of <span>BCL11B </span>and <span>Satb2</span> 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, <i>via</i> CB₁ receptor, promotes mouse and human deep layer cortical neuron development.</p>