EVIDENCE FOR DIFFERENTIAL EFFECTS OF Δ9-THC (Δ9-TETRAHYDROCANNABINOL) ON PRE- VERSUS POST-IMPLANTATION EMBRYONIC STEM CELLS

Recreational marijuana use is becoming increasingly widespread. With potential implications for fertility and embryonic development, we set out to evaluate the impact of THC exposure on the pre- and post-implantation embryo through embryonic stem cell proliferation, mitochondrial function and metabolism.

Mouse embryonic stem cells (ESCs) can be differentiated into epiblast-like cells (EpiLCs) using known in vitro methods via growth factors and cytokines. ESCs recapitulate important features of pre-implantation stem cells; EpiLCs model post-implantation features. ESCs and EpiLCs were exposed to a range of Δ9-THC concentrations: 0-100μM over 48 hours. Cell proliferation and viability were assessed via Trypan blue and Countess II FL Automated Cell Counter. The cells were exposed to WST-1 and analyzed by ELISA reader to measure wavelength absorption as a proxy for oxidative phosphorylation. The live cells were stained with a MitoTracker dye to label the mitochondria. Changes in membrane potential were measured whereby increases in membrane potential represented mitochondrial disruption and early stages of cell apoptosis. Comparative analyses between the untreated versus treated cells were performed with unpaired t-tests.

In response to 10nM and 100nM Δ9-THC concentrations, ESCs demonstrated a biphasic dose-dependent proliferation response: low concentration THC increased proliferation, whereas high-dose THC increased apoptosis. These effects were not observed in EpiLCs. We demonstrated these biphasic results are due in part to differential effects on membrane potential and oxidative phosphorylation.

Compared to untreated cells, Δ9-THC at low doses significantly increases proliferation in ESCs but not EpiLCs. We demonstrate early evidence for metabolic differences underlying this unique proliferation dependence. Given ESCs represent pre-implantation cells, while EpiLCs model post-implantation cells, these data suggest an intriguing hypothesis that implantation may trigger a shift in endocannabinoid metabolism. Future work will be addressed toward clarifying these effects.

Although the impact of Δ9-THC on early developmental differentiation and programming is unknown, it is possible that Δ9-THC exposure may disrupt the metabolism and epigenetic machinery in germ cells. Given that cannabis is the most widely illicit drug used in the world, with increasing consumption in women of reproductive age, our work is relevant to understanding the basic action of cannabis on embryonic stem cells. Future research should investigate the transgenerational effect of marijuana use from in utero exposure.