3.7. Cannabis affects cell proliferation and viability but not cell differentiation
Cell proliferation was evaluated by immunostaining cells for the specific Ki67 marker (Fig. 6D-E) and there was an increase in the number of proliferative ON cells from cannabis users relative to the controls (t = 4.212, p = 0.0029, Fig. 6B). This increase was consistent with the proteomic analysis in which there was an up-regulation of cell growth and proliferation proteins in cells from cannabis users (Fig. 4C) and proteins involved in cell cycle regulation (Supplementary Fig. 1). Despite the increase in proliferation, the total number of cells in culture was no different between the two groups (t = 0.393, p = 0.704, Fig. 6C).
To evaluate whether the changes in cell proliferation may affect differentiation, the expression of the NeuN neuronal marker and the GFAP astrocyte marker was assessed (Fig. 6G-J). After 7 days in culture, 40.22% of the cells were NeuN+ in both groups, whereas no GFAP+ cells were detected, indicating a high degree of mature cells expressing a neuronal, but not a glial marker. However, no differences were observed between groups in NeuN+ labelling (t = 0.0889 p = 0.9313, Fig. 6F).
Finally, proteomic results indicated a potential down-regulation of cell death, based on the differential expression of the proteins identified (Fig. 4B). We observed a significant decrease in the proportion of Annexin V+/PI- and Annexin V+/PI+ cells derived from cannabis users relative to the controls, indicating a decrease in both early and late apoptosis in ON cells from cannabis users (t = 2.975, p = 0.0177 and t = 2.827, p = 0.0222 respectively: Fig. 6K-O), consistent with the proteomic data.