A Complete Endocannabinoid Signaling System Modulates Synaptic Transmission between Human Induced Pluripotent Stem Cell-Derived Neurons

Abstract

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.

Fig. 1.

hiPSC-derived neuronal cultures exhibit synaptically driven [Ca^2+] _i spiking. (A) hiPSC-derived neuronal cultures expressing mCherry from the GFAP promoter (magenta) and GFP from the synapsin promoter (green). Scale bar, 50 µm. (B) Representative image of hiPSC-derived cultures in Calcium 6 dye (left) and ROIs derived from this image using threshold-based segmentation in ImageJ (right). Scale bar, 50 µm. (C) Individual [Ca²⁺]_i traces representing ΔF/F₀ for each ROI in (B). Scale bar, 30 seconds. (D) Representative traces show mean [Ca^2+] _i (solid line) from a single field of hiPSC-derived neuronal cells (S.D. denoted by blue shading) in 0.1 mM Mg²⁺ buffer containing H₂O vehicle (top), 10 µm tetrodotoxin (TTX, middle), or pretreated (18 hours) with 2.5 µm tetanus toxin (TeNT, bottom). Scale bars: horizontal, 30 seconds; vertical, 1.0 ΔF/F₀. Bar graph summarizes Ca²⁺ spiking activity (events per minute) in cultures treated with H₂O vehicle (CTL), TTX, or TeNT as depicted in traces. *P < 0.05 relative to CTL. Kruskal-Wallis test with Dunn’s multiple comparisons correction, P = 0.024 for TTX and 0.013 for TeNT relative to vehicle, n = 3 to 4 wells per treatment.

Fig. 2.

hiPSC-derived neuronal cultures form a glutamatergic synaptic network. (A) Schematic shows drug treatments sequence for this and following figures. (B) Representative traces show mean [Ca²⁺]_i (solid line) for a single field of hiPSC-derived neuronal cells (S.D. denoted by blue shading) in 0.1 mM Mg²⁺ buffer before (baseline epoch) and after (treated epoch) the addition of vehicle (H₂O, top), 100 µM CNQX (middle), or 10 µM MK801 (bottom). Scale bars: horizontal, 30 seconds; vertical, 1.0 ΔF/F₀. (C) Bar graph summarizes the change in [Ca²⁺]_i spiking activity (percentage of change in AUC) before and after adding vehicle (H₂O for CNQX and DMSO for MK 801), 100 µM CNQX, or 10 µM MK 801. **P < 0.01 relative to vehicle control; ***P < 0.001 relative to vehicle. Data are presented as mean ± S.D. Student’s t test: t = 4.9 and P = 0.002 for CNQX relative to H₂O; t = 7.0 and P = 0.0004 for MK 801 relative to DMSO; n = 4 to 5 wells per condition.

Fig. 3.

Cannabinoid agonists inhibit synaptic activity between hiPSC- derived neurons via CB₁R. (A–C) Representative traces show mean (solid) [Ca²⁺]_i (ΔF/F₀) of a single field of hiPSC-derived neuronal cells (S.D. denoted by blue shading) in 0.1 mM Mg²⁺ buffer before and after the addition of 300 nM Win-2 (A), 3 µM 2-AG (B), or 1 µM Win-2 in the presence of 100 nM NESS 0327 (C). Scale bars: horizontal, 30 seconds; vertical, 1.0 ΔF/F₀. (D) Concentration-response curves for Win-2 (circles) and 2-AG (triangles) were fit with a logistic equation of the form: percentage of change in AUC = A₁ + [(A₂-A₁)/(1 + 10^(logx₀-x)p)], where x₀ = EC₅₀, x = log[drug], A₁ = percentage of change in the absence of drug, A₂ = percentage of change at a maximally effective drug concentration, and p = slope factor. The following values were calculated using a nonlinear, least-squares curve-fitting program: A₁ = −25% for Win-2 and −21% for 2-AG; A₂ = -84% for Win-2 and −86% for 2-AG; EC₅₀ = 48 ± 13 nM for Win-2 and 2.0 ± 0.6 µM for 2-AG; p = −3.0 ± 1.4 for Win-2 and −2.0 ± 1.0 for 2-AG. n = 4–6 wells per concentration. (E) Bar graph summarizes change in [Ca²⁺]_i spiking induced by 1 µM Win-2 in wells treated with vehicle (0.1% ethanol, left) or 100 nM NESS 0327 (right). Data are presented as mean ± S.D. **P < 0.01. Welch’s t test, t_(6.4) = 5.4, P = 0.0014, 6 wells per condition.

Fig. 4.

Δ⁹-THC acts as a partial agonist to inhibit synaptic transmission in hiPSC-derived neuronal cultures. (A) Representative traces show mean (solid line) [Ca²⁺]_i (ΔF/F₀) of a single field of hiPSC-derived neuronal cells (S.D. denoted by blue shading) in 0.1 mM Mg²⁺ buffer before and after the addition of 1 µM Δ⁹-THC in the absence (upper) or presence of 100 nM NESS 0327 (lower). Scale bars: horizontal, 30 seconds; vertical, 1.0 ΔF/F₀. (B) Concentration-response curve for Δ⁹-THC was fit with a logistic equation of the form percentage of change in AUC = A₁ + [(A₂-A₁)/(1 + 10^(logx₀-x)p)], where x₀ = EC₅₀, x = log[Δ⁹-THC], A₁ = percentage of change in the absence of drug, A₂ = percentage of change at a maximally effective drug concentration, and p = slope factor. The following values were calculated using a nonlinear, least-squares curve-fitting program: A₁ = −17 ± 11%; A₂ = −47 ± 14%; EC₅₀ = 1.4 ± 1.9 µM; p = −0.9 ± 1.2; n = 4–7 wells per concentration. (C) Representative traces show mean (solid line) [Ca²⁺]_i (ΔF/F₀) of a single field of hiPSC-derived neuronal cells (S.D. denoted by blue shading) in 0.1 mM Mg²⁺ buffer before and after the addition of 1 µM Win-2 in the presence of vehicle (0.01% ethanol, top) or 1 µM Δ⁹-THC (bottom). Scale bars: horizontal, 30 seconds; vertical, 1.0 ΔF/F₀. (D) Bar graph shows change in [Ca²⁺]_i spiking relative to baseline (AUC) after addition of 1 µM Δ⁹-THC in wells treated with vehicle or 100 nM NESS 0327. Data are presented as mean ± S.D. Unpaired Student’s t test, t₍₁₀₎ = 2.3, *P < 0.05, n = 6 wells per condition. (E) Bar graph shows change in [Ca²⁺]_i spiking relative to baseline (AUC) after addition of 1 µM Win-2 in wells treated with vehicle or Δ⁹-THC. Data are presented as mean ± S.D. Unpaired Student’s t test, t_(5.5) = 18.8, **P < 0.0001, n = 6 wells per condition. Veh, vehicle.

Fig. 5.

eCB GRAB sensor imaging in hiPSC-derived neuronal cultures. (A) Left: GRAB_eCB2.0 fluorescence (ΔF/F₀) in cells treated with 300 nM Win-2. Scale bar, 10 µm. Right: enlarged images of the boxed region at baseline (top) and after the addition of 1 µM carbachol (middle) and 300 nM Win-2 (bottom). Scale bar, 10 µm. (B) Schematic showing the mechanism of carbachol-triggered 2-AG synthesis. (C) Time course of GRAB_eCB2.0 fluorescence (ΔF/F₀) for the ROI highlighted in (A) expressed as a percentage of the response in saturating Win-2 (mean of final 40 seconds of recording). Arrowheads mark the addition of 1 µM carbachol (Cch) and 300 nM Win-2. DAGL, diacylglycerol lipase; mAChR, muscarinic acetylcholine receptor; PIP₂, phosphatidylinositol bisphosphate; PLC-β, phospholipase C-β.

Fig. 6.

hiPSC-derived neuronal cultures synthesize eCBs via DAG lipase. (A) Time courses showing mean GRAB_eCB2.0 fluorescence (ΔF/F₀, expressed as a percentage of the Win-2 response) from representative wells pretreated for 1 hour with vehicle (0.1% DMSO) or 10 or 30 nM DO34. Arrowheads mark the addition of 1 µM carbachol (Cch) and 300 nM Win-2. n = 4 ROIs per well. (B) Bar graph summarizes peak carbachol-evoked GRAB_eCB2.0 fluorescence (ΔF/F₀, expressed as a percentage of the Win-2 response) in wells pretreated for 1 hour with vehicle (0.1% DMSO), 10 nM DO34, or 30 nM DO34. Data are presented as mean ± S.D. *P < 0.05 relative to vehicle. Brown-Forsythe ANOVA test with Dunnet’s T3 multiple comparisons test, F_{(2.0, 3.1)} = 17.45, P = 0.045 for 10 nM DO34 and 0.040 for 30 nM DO34 versus vehicle. n = 4 to 5 wells; each well is the average of 4 ROIs.

Fig. 7.

hiPSC-derived neuronal cultures metabolize 2-AG via MAG lipase. (A) Traces showing average GRAB_eCB2.0 fluorescence from representative wells pretreated with vehicle or 1 µM JZL 184. Arrowheads show addition of 1 µM carbachol (Cch), washout of carbachol (wash), and addition of 300 nM Win-2. Each trace is the average of 4 ROIs from a single well. Scale bars: horizontal, 30 seconds; vertical, 20% of saturating Win-2 response (ΔF/F₀). (B) Time course showing the decay of carbachol-evoked GRAB_eCB2.0 fluorescence after washout of carbachol (ΔF/F₀, expressed as a percentage of the peak fluorescence on a log scale) in cells pretreated for 1 hour with vehicle (0.1% DMSO, filled circles) or 1 µM JZL 184 (open squares). One JZL 184 well decayed too slowly to calculate a time constant and is not included. n = 6 wells for vehicle and 5 wells for JZL 184. Data are presented as mean ± S.D. Curves were fit with a linear regression. (C) Bar graph shows time constants for GRAB_eCB2.0 fluorescence decay calculated from the individual recordings averaged in (B). Individual curves were fit with an exponential decay function constrained to a y-intercept of 100 and an asymptote approaching 0. Data are presented as mean ± S.D. **P < 0.01. Unpaired Student’s t test, t₍₉₎ = 4.5, n = 6 wells for vehicle and 5 wells for JZL 184.

Fig. 8.

Carbachol (Cch)-evoked 2-AG production is independent of [Ca²⁺]_i. (A) Top: representative carbachol-evoked [Ca²⁺]_i traces from wells pretreated for 30 minutes with vehicle (0.1% DMSO) or 1 µM thapsigargin in the absence or presence of Ca²⁺as indicated. Horizontal bar, 30 seconds. Bottom: representative traces showing GRAB_eCB2.0 fluorescence in wells pretreated as indicated. Horizontal bar, 30 seconds. (B) Bar graph summarizes peak increases in [Ca²⁺]_i evoked by 1 µM carbachol in the absence or presence of 1 µM thapsigargin. Recordings were performed in the absence (nominally Ca²⁺ free) or presence of 1.3 mM extracellular Ca²⁺ as indicated. n = 6 wells per condition. Data are presented as mean ± S.D. Brown-Forsythe ANOVA (F_{(2, 8.4)} = 12.9, P = 0.0028) with Dunnet’s T3 multiple comparisons test **P < 0.01, relative to vehicle + Ca²⁺ + carbachol. (C) Peak carbachol-evoked GRAB_eCB2.0 fluorescence in cells treated as indicated. n = 4 wells per condition; each well value is the average of 4 ROIs. Data are presented as mean ± S.D. Brown-Forsythe ANOVA F_{(2, 8.9)} = 0.3, P = 0.75. Thaps, thapsigargin; Veh, vehicle.

Fig. 9.

Comparison of Win-2 to JZL184–induced desensitization of CB₁R-mediated inhibition of synaptic activity. (A) Timeline of pretreatment and imaging for desensitization experiments. (B) Representative traces show mean (solid line) [Ca²⁺]_i (ΔF/F₀) of a single field of hiPSC-derived neuronal cells (S.D. denoted by blue shading) in 0.1 mM Mg²⁺ buffer before and after the addition of 1 µM Win-2 (arrowheads) in cells pretreated for 7 days with either vehicle (0.1% DMSO, top), 1 µM Win-2 (middle), or 1 µM JZL 184 (bottom). Scale bars: horizontal, 30 seconds; vertical, 1.0 ΔF/F₀. (C) Plot shows inhibition of [Ca²⁺]_i spiking activity produced by 1 µM Win-2 in cells pretreated for the indicated times with vehicle (0.1% DMSO, black circles), 1 µM Win-2 (red squares), or 1 µM JZL 184 (green triangles). Data are presented as mean ± S.D. n = 6 wells per condition. *P < 0.05 and ****P < 0.0001 relative to vehicle; †P < 0.05; ††P < 001; ††††P < 0.0001 relative to Win-2. Two-way repeated measures ANOVA with Tukey’s multiple comparisons test. Pretreatment time F_{(3, 59)} = 16.1, P < 0.0001; pretreatment drug F_{(2, 59)} = 67.2, P < 0.0001, interaction F_{(6, 59)} = 4.3, P = 0.0012.

Fig. 10.

CB₁R activation inhibits [Ca²⁺]_i spiking in hiPSC-derived neuronal cultures from multiple sources. (A) Individual [Ca²⁺]_i traces representing ΔF/F₀ for spontaneously active ROIs in a cell culture produced from cells obtained from BrainXell. Recording was performed in 0.1 mM Mg²⁺ buffer, and cells were treated with 1 µM Win-2 at the time indicated by the arrow. Scale bar: horizontal, 30 seconds; vertical, 1.0 ΔF/F₀. (B) Representative traces show mean (solid line) [Ca²⁺] _i (ΔF/F₀) and S.D. (denoted by blue shading) from a single field of hiPSC-derived neuronal cells (obtained from Applied StemCell) in 0.1 mM Mg²⁺ buffer treated with 1 µM Win-2 at the time indicated by the arrow. Scale bars: horizontal, 30 seconds; vertical, 1.0 ΔF/F₀. (C) Bar graph summarizes change in [Ca²⁺]_i spiking induced by 1 µM Win-2 in wells treated with vehicle (0.1% ethanol, open bars) or 100 nM NESS 0327 (filled bars) from cells obtained from BrainXell or Applied StemCell as indicated. Data are presented as mean ± S.D. *P < 0.05; **P < 0.01, unpaired Student’s t test. For BrainXell hiPSCs, t₍₁₀₎ = 3.8, P = 0.003, 6 wells per condition. For Applied StemCell hiPSCs, t₍₆₎ = 2.3, P = 0.049, 4 wells per condition. Veh, vehicle.