A complete endocannabinoid signaling system modulates synaptic transmission between human induced pluripotent stem cell-derived neurons

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{plus minus}13 nM) by the efficacious agonist Win55,212-2 ([R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrolol [1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate]) and by the endogenous ligand 2-arachidonoyl glycerol (2-AG; EC₅₀=2.0{plus minus}0.6 µM). The effects of Win55212-2 were blocked by a CB₁ receptor-selective antagonist. Δ⁹-tetrahydrocannabinol acted as a partial agonist, maximally inhibiting synaptic activity by 47{plus minus}14% (EC₅₀=1.4{plus minus}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 JZL-184 (4-[Bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester). Win 55,212-2 markedly desensitized CB₁ receptor function following a 1 d exposure, while desensitization was incomplete following 7 d 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. 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.