Fig. 5: The endocannabinoid agonist WIN 55,212-2 selectively depletes human-derived dorsal forebrain organoids of neurons.

a Schematic of neocortical neurogenesis pulse-chase assay. Neocortical neurogenesis was evaluated within human-derived dorsal forebrain organoids via the adaptation of a BrdU pulse-chase assay [70]. Briefly, at the time of treatment commencement, dorsal forebrain organoids were pulsed for 24 h with BrdU. Following this, BrdU was removed and washed out and cultures underwent continuation of their allotted treatment to their endpoints. Organoids were subsequently processed (fixed, dehydrated, cryosectioned, mounted) before being immunostained and imaged for newborn (BrdU + ; red) neurons (MAP2 + ; green) via laser-scanning confocal microscopy. b Representative whole organoid images of enviromimetic treated organoids. Representative whole organoid images for each treatment group are shown as insets. Note that in all groups there was widespread and robust expression of both BrdU (red) and MAP2 (green) across the entire organoid, except those treated with WIN 55,212-2. To emphasize this, MAP2-isolated channels are presented in gray scale below merged images. c–e Depletion of newborn and total neurons in WIN 55,212-2 treated organoids. To determine if our various treatments influenced neuron numbers, we quantified the number of newborn neurons MAP2⁺ green cells with BrdU⁺ red nuclei, see (d) and total neurons MAP2⁺ green cells with DAPI⁺ blue nuclei, see (e) within cortical fields. The only group to exhibit a significant difference in both newborn and total neuron numbers within the developing cortical plates of dorsal forebrain organoids was the cannabinoid agonist WIN 55,212-2. More specifically, we identified that WIN 55,212-2 treatment reduced newborn neurons by 81.23% and total neurons by 75%. When combined with our single-cell DNA content and DNA-damage cell death flow cytometry panels presented in Fig. 4, this data cumulatively confirms that WIN 55,212-2 is acutely neurotoxic and developmentally disruptive within human-derived dorsal forebrain organoids. For all panels, n = 3 iPSC donors x n = 7 treatment groups x n = 3–4 organoid replicates per donor/condition yielded a total n of = 18–22 cortical fields per condition. **** denotes p < 0.0001. Bar graphs represent mean ± SEM. Scale bar in b = 100 μm and c = 40 μm.