Figure 6. PGCLCs deriving from ESCs and EpiLCs exposed to 100 nM of Δ9-THC proliferate.

(A) Diagram illustrating Δ9-THC exposure scheme and experimental strategy. (B) Representative flow contour plots showing distribution of live-gated events, gating strategy for Stella:CFP versus Blimp1:mVenus and percentages of cells in each subpopulations for ESCs and EpiLCs exposed to the different doses of Δ9-THC indicated. DN: double negative, SP: single positive, DP: double positive subpopulations. (C) The percentage of events in the gates associated to each subpopulation was normalized to the one measured in the mock-treated condition. Median and associated errors were plotted in whisker boxplots independently for each subpopulation. (D) Histograms showing CellTrace Yellow staining profile of cells arising from ESCs and EpiLCs exposed to the different doses of Δ9-THC indicated. The Y-axis represents the average percentage of cells in each category of subpopulations undividing (purple), undergoing 1 division (blue), 2 divisions (green) or 3 divisions (orange). One representative experiment out of three is represented. Statistical significance: *(p<0.05), **(p<0.01), ***(p<0.001), ****(p<0.0001).

Figure 6—figure supplement 1. PGCLCs gating and sorting strategy.

(A) Representative flow contour plots showing distribution of events and gating based on embryoid bodies dissociation. (B and C) Representative flow contour plots to isolate singlets based on width to height ratios on the side scatter and front scatter, respectively. (D) Gating strategy for Stella:CFP versus Blimp1:mVenus on the negative control, corresponding to embryoid bodies obtained in an induction medium without cytokines and BMPs (GK15 only). (E) Gating strategy for Stella:CFP versus Blimp1:mVenus on mock-treated cells, corresponding to embryoid bodies obtained in an induction medium complemented with cytokines and BMPs. DN: double negative, SP: single positive, DP: double positive subpopulations.

Figure 6—figure supplement 2. Male PGCLCs deriving from ESCs and EpiLCs exposed to 100 nM of Δ9-THC proliferate.

(A) Diagram illustrating Δ9-THC exposure scheme and experimental strategy. (B) Representative flow contour plots showing distribution of live-gated events, gating strategy for Stella:CFP versus Blimp1:mVenus and percentages of cells in each subpopulations for ESCs and EpiLCs exposed to 100 nM of Δ9-THC. DN: double negative, SP: single positive, DP: double positive subpopulations. (C) The percentage of events in the gates associated to each subpopulation was normalized to the one measured in the mock-treated condition. Median and associated errors were plotted in whisker boxplots independently for each subpopulation. At least three independent biological repeats with three technical replicates (N=3, n=3). Statistical significance: *(p<0.05).

Figure 6—figure supplement 3. No residual Δ9-THC is detected in day 5 embryoid bodies.

Intracellular levels of ∆9-THC were quantified by mass spectrometry in EpiLCs on the day of aggregate formation and in day 5 embryoid bodies (referred as to ‘EpiLCs’ and ‘PGCLCs’). Histograms show the median and associated errors of two independent quantifications. Statistical significance: ****(p<0.0001), one-way ANOVA with post-hoc Tukey HSD.