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. 2021 Jun 17;11(6):565. doi: 10.3390/jpm11060565

Figure 4.

Figure 4

Promoter-dependent iC9-mediated selective ablation of iPSCs. (a) Lentiviral vectors using two different promoters, i.e., either the EOS (LV EOS.iC9) or the human Oct4SRE (LV Oct4SRE.iC9), for pluripotency-specific expression of the iC9, Kat2S and ΔCD19 transgenes. LTR = long terminal repeat; ∆U3 = deleted enhancer/promoter region; R = repeat region within the LTR; Ψ = PSI, packaging signal; PBS = primer binding site; wPRE = woodchuck post-transcriptional regulatory element. (b) Percentage of viability relative to non-treated control cells (Ntc) as determined by CellTiterGlo® 2.0 cell viability assay. Unmodified iPSCs (iPSC ctrl) and transgenic iPSCs (LV ctrl, LV EOS.iC9, LV Oct4SRE.iC9) were treated for 24 h with the dimerizer AP20187 at the indicated concentrations, with vehicle (EtOH) only or left untreated (Ntc). N = 5. (c) Microscopy images of the indicated iPSC cultures treated with vehicle (EtOH) or with 0.1 nM AP20187 upon 24 h of treatment. Scale bar: 100 µm. (d) Percentage of viability of iMAC relative to non-treated cells as determined by CellTiterGlo® 2.0 cell viability assay. Unmodified induced macrophages (iMAC ctrl) and transgenic iMAC (LV EOS.iC9, LV Oct4SRE.iC9, LV CAGs.iC9) were treated for 24 h with the dimerizer AP20187 at the indicated concentrations with the vehicle (EtOH) only or left untreated (Ntc). N = 3. (e) Tumor volume, determined with a caliper weekly, at the indicated time points after injection of LV Oct4SRE.iC9-transduced iPSCs into NSG mice. AP20187 or vehicle (AP20187-buffer) was administered immediately upon iPSC injection. Each data point represents one teratoma. N = 3–7. Arrows indicate the time points of AP20187/vehicle (AP20187-buffer) treatment. Cystic tumors (grey triangles) were excluded from the analysis. (f) Tumor volume at the indicated time points after injection of LV Oct4SRE.iC9-transduced iPSCs into NSG mice. AP20187 or AP20187-buffer was administered upon emergence of palpable tumors. Each data point represents one teratoma. N = 5–7. Arrows indicate the time points of AP20187/vehicle (AP20187-buffer) treatment. Cystic tumors (grey triangles) were excluded from the analysis. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.