hiPSC-Based Model of Prenatal Exposure to Cannabinoids: Effect on Neuronal Differentiation.

. 2020 Jul 6;13:119.

doi: 10.3389/fnmol.2020.00119. eCollection 2020.


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Cláudia C Miranda et al. Front Mol Neurosci. .


Phytocannabinoids are psychotropic substances ofcannabis with the ability to bind endocannabinoid (eCB) receptors that regulate synaptic activity in the central nervous system (CNS). Synthetic cannabinoids (SCs) are synthetic analogs of Δ9-tetrahydrocannabinol (Δ9-THC), the psychotropic compound of cannabis, acting as agonists of eCB receptor CB1. SC is an easily available and popular alternative to cannabis, and their molecular structure is always changing, increasing the hazard for the general population. The popularity of cannabis and its derivatives may lead, and often does, to a child’s exposure to cannabis both in utero and through breastfeeding by a drug-consuming mother. Prenatal exposure to cannabis has been associated with an altered rate of mental development and significant changes in nervous system functioning. However, the understanding of mechanisms of its action on developing the human CNS is still lacking. We investigated the effect of continuous exposure to cannabinoids on developing human neurons, mimicking the prenatal exposure by drug-consuming mother. Two human induced pluripotent stem cells (hiPSC) lines were induced to differentiate into neuronal cells and exposed for 37 days to cannabidiol (CBD), Δ9-THC, and two SCs, THJ-018 and EG-018. Both Δ9-THC and SC, at 10 μM, promote precocious neuronal and glial differentiation, while CBD at the same concentration is neurotoxic. Neurons exposed to Δ9-THC and SC show abnormal functioning of voltage-gated calcium channels when stimulated by extracellular potassium. In sum, all studied substances have a profound impact on the developing neurons, highlighting the importance of thorough research on the impact of prenatal exposure to natural and SC.

Keywords: CBD; EG-018; THJ-018; hiPSC; neuronal differentiation; phytocannabinoids; synthetic cannabinoids; Δ9-THC.


Figure 1

Figure 1

Efficient neural differentiation of hiPSCs and the effect of cannabinoid exposure. (A) qRT-PCR analysis of neural progenitor (PAX6) and neuronal (MAP2) mRNA expression levels relative to GAPDH at indicated timepoints. Data were analyzed by unpaired t-test, *p < 0.05, **p < 0.01; error bars represent standard error of the mean (SEM). (B) qRT-PCR analysis of PAX6 and MAP2 mRNA levels in untreated vs. vehicle-treated (0.01% EtOH) cultures showing no significant differences by unpaired t-test. Data in panels (A,B) were obtained from four independent experiments using iPSC6.2 cells. (C) Immunofluorescence for neural progenitor, neural and glial markers at different timepoints showing efficient neural commitment and differentiation of hiPSCs. Scale bars in panels (i,i’), 100 μm. Scale bars in panels (ii–vii), 50 μm. (D) Immunofluorescence at day 30 for neural progenitor marker PAX6 and neuron-specific microtubule-associated protein MAP2 in untreated (i, vii), vehicle-treated (ii, viii), and exposed to cannabinoids from day 19 to day 30 cultures (iii–vi, ix–xii), in two different iPSC lines, iPCS6.2 (male donor) and F002.1A.13 (female donor). Scale bars: 50 μm.

Figure 2

Figure 2

Effect of cannabinoid exposure on day 30 and 56 of neural differentiation. (A) Immunofluorescence for newborn neuronal marker HuC/D and apoptosis marker pCASPASE3 (pCASP3) at day 30 showing an increase in HuC/D staining and apoptotic cells in s cannabinoid-treated cultures. Scale bars: 15 μm. (B) Immunofluorescence for neuronal (MAP2) and synaptic protein synaptophysin (SYN) in cultures continuously exposed to cannabinoids from day 19 to day 56. Scale bars: 15 μm. (C) Quantification of PAX6+(i), HuC/D+(ii) and pCASP3+(iii) cells at day 30, relative to DAPI. Results for three independent experiments. (D) Quantification of the ratio of fluorescence intensity of SYN and MAP2 in day 56 cultures (i), and of integrated fluorescence density for GFAP (ii). Data from three independent experiments, 3–10 images per condition. Data in (C) and (D) analyzed by unpaired t-test; *p < 0.05, **p < 0.01; error bars represent SEM. Tukey’s range test was applied to determine outlier data points (open circles). (E) Immunofluorescence for glial (GFAP) marker and MAP2 in cultures continuously exposed to cannabinoids from day 19 to day 56. Scale bars: 15 μm.

Figure 3

Figure 3

Functional assessment of cannabinoid-treated cultures on day 56. (A) Single-cell calcium imaging (SCCI) analysis of day 56 cultures showing abnormal response to KCl and histamine stimuli by Δ9-THC, EG-018, and THJ-018-treated neuronal cells. (B) Summary of SCCI analyses presented as percentage of responding and non-responding cells for KCl and histamine stimulation. The timepoint of response corresponds to the peak seen in the graph in panel (A), and the ratio of fluorescence value for this timepoint over baseline level >1 was considered as a response. For EG-018 and THJ-018 conditions, the last time point before washing was used to calculate the response to histamine stimulation. (C) Hierarchical clustering illustrates relative expression levels of different genes at day 30 and 56 of neural differentiation. PAX6, MAP2, GAD67, VGLUT1, CNR1, and CNR2 were analyzed by qRT-PCR. Rows are centered; unit variance scaling is applied to rows. Rows are clustered using correlation distance and average linkage. Corresponding qRT-PCR data are presented in Supplementary Figure S2. (D) Single-cell calcium imaging. Representative ratio images for different culture conditions on day 56. Images were taken immediately after cells received the indicated stimulus (KCl or histamine).

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