A Complete Endocannabinoid Signaling System Modulates Synaptic Transmission between Human Induced Pluripotent Stem...

The endocannabinoid system (ECS) modulates synaptic function to regulate many aspects of neurophysiology. It adapts to environmental changes and is affected by disease. Thus, the ECS presents an important target for therapeutic development. Despite recent interest in cannabinoid-based treatments, few preclinical studies are conducted in human systems. Human induced pluripotent stem cells (hiPSCs) provide one possible solution to this issue. However, it is not known if these cells have a fully functional ECS. Here, we show that hiPSC-derived neuron/astrocyte cultures exhibit a complete ECS. Using Ca²⁺ imaging and a genetically encoded endocannabinoid sensor, we demonstrate that they not only respond to exogenously applied cannabinoids but also produce and metabolize endocannabinoids. Synaptically driven [Ca²⁺]_i spiking activity was inhibited (EC₅₀ = 48 ± 13 nM) by the efficacious agonist [R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrolol [1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate] (Win 55,212-2) and by the endogenous ligand 2-arachidonoyl glycerol (2-AG; EC₅₀ = 2.0 ± 0.6 µm). The effects of Win 55212-2 were blocked by a CB₁ receptor-selective antagonist. Δ⁹-Tetrahydrocannabinol acted as a partial agonist, maximally inhibiting synaptic activity by 47 ± 14% (EC₅₀ = 1.4 ± 1.9 µm). Carbachol stimulated 2-AG production in a manner that was independent of Ca²⁺ and blocked by selective inhibition of diacylglycerol lipase. 2-AG returned to basal levels via a process mediated by monoacylglycerol lipase as indicated by slowed recovery in cultures treated with 4-[Bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester (JZL 184). Win 55,212-2 markedly desensitized CB₁ receptor function following a 1-day exposure, whereas desensitization was incomplete following 7-day treatment with JZL 184. This human cell culture model is well suited for functional analysis of the ECS and as a platform for drug development. SIGNIFICANCE STATEMENT: Despite known differences between the human response to cannabinoids and that of other species, an in vitro human model demonstrating a fully functional endocannabinoid system has not been described. Human induced pluripotent stem cells (hiPSCs) can be obtained from skin samples and then reprogrammed into neurons for use in basic research and drug screening. Here, we show that hiPSC-derived neuronal cultures exhibit a complete endocannabinoid system suitable for mechanistic studies and drug discovery.