Cannabinoids affect differentiation of human iPSC-derived neurons

Abstract

Event Abstract Back to Event Cannabinoids affect differentiation of human iPSC-derived neurons Tiago C. Barata1, Claudia Miranda1, Sandra Vaz2, Carla S. Ferreira3, Alexandre Quintas3 and Evguenia P. Bekman1, 2* 1 Institute for Biotechnology and Bioengineering, Portugal 2 Institute of Molecular Medicine, Faculty of Medicine, University of Lisbon, Portugal 3 Centro de investigação interdisciplinar Egas Moniz, Portugal Phytocannabinoids are psychotropic substances found in cannabis that bind to the endocannabinoid receptors regulating a variety of physiological processes in human body, including synaptic activity in the central nervous system and metabolic effects in the peripheric nervous system, among many others (1,2). Synthetic cannabinoids emerged as popular alternative to cannabis. Most of these substances are synthetic analogues of Δ9-THC, the psychotropic compound of cannabis, usually with higher affinity to the endocannabinoid receptor CB1 and/or a slower dissociation kinetics, capable to elicit a stronger and long-lasting effect on brain cells. Synthetic cannabinoids are easily available, and their molecular structure is always changing, increasing hazard for general population. The popularity of cannabis and its derivatives may lead, and often does, to child’s exposure to cannabis both in utero and through breastfeeding by a drug-consuming mother. Prenatal exposure to cannabis has been associated with higher risk of newborn morbidity (2,3), altered rate of mental development and significant changes in nervous system functioning (4,5). However, direct evidence that these effects are mediated through the binding of cannabinoids to endocannabinoid receptors is still lacking. Thus, it is paramount to better understand the psychoactive effects of natural and synthetic cannabinoids, on the developing human brain. We conveyed a pilot study in which human induced pluripotent stem cells (hiPSCs) were induced into neural differentiation and treated with a non-psychotropic component of cannabis, cannabinol, known to bind the CB2 receptor, and two synthetic Δ9-THC analogues, THJ and EG-018. The goal of this study was to test the hypothesis that exposure of developing human neural cell to cannabinoids influences neuronal differentiation. To approach this, hiPS cells were induced into in vitro neural commitment and differentiation using modified dual SMAD inhibition protocol (6) to allow for functional maturation and exposed from day 19 to day 32 of differentiation to EG-18, THJ and cannabinol directly supplemented to the culture medium. Cultures were analysed at days 32, 56 and 80 of differentiation for the presence of neural progenitor, neuronal and glial markers by immunofluorescence and additionally, functional analysis of the cultured cells was performed at day 56 by single cell calcium imaging (SCCI). The data revealed striking differences between untreated control and treated cells. Cultures treated with cannabinol exhibited a decrease in PAX6+ progenitors by day 32 and almost total depletion in GFAP+ progenitor/glial cells on later stages. MAP+ neuronal cells showed shorter non-ramified neurites and very few functional neurons capable to respond to KCl stimulus were found in this condition indicating a strong negative effect of this substance on neuronal differentiation. Contrary to the cannabinol, cultures treated with synthetic cannabinoids demonstrated presence of higher proportion of mature neurons in both cultures at day 56, indicating that both synthetic cannabinoids are able to promote neuronal differentiation also increase the length and branching of neurites in both MAP+ neurons and GFAP+ glia. However, SCCI analysis showed that the functionality of neural cells grown in these conditions appears to be compromised. Further studies are needed to better understand the observed functional impairment of cannabinoid-treated neurons. Overall these data clearly show that all three compounds used in this study strongly influence the process of neuronal differentiation and thus, most likely are able to impact on the development of human CNS. Acknowledgements Authors thank Cooperativa de Ensino Superior Egas Moniz, CRL for funding this research