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

The endocannabinoid (eCB) system, via the 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 during 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 the CB1 receptor, promotes mouse and human deep layer cortical neuron development. This work was supported by the European Regional Development Fund ‘A way to achieve Europe’ (PI18-00941 to I.G.-R., RTI2018-095311-B-100 to M.G., BFU2017-83292-R to J.S.-P. and RTI2018-101663-B-100 to I.L.). J.P.-L., J.A. and S.S.-S., were supported by FPU, FPI and PFIS program fellowships, respectively, from the Ministerio de Educación, Cultura y Deporte, Ministerio de Ciencia and Ministerio de Sanidad. S.S.-S. was also supported by the Fondo Social Europeo-La Iniciativa sobre Empleo Juvenil (YEI) (CT101/18-CT102/18PEJD-2018-PRE/BMD-7933). A.d.S.-Q., D.G.-R. and R.M. were supported by the Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Fundación Tatiana Pérez de Guzmán el Bueno and European Regional Development Fund (BFU2017-83292-R), respectively. Sí