Informed consent forms.

(ZIP)</p

scRNA-seq analysis of purified OSR1(+)SIX2(+) induced NPCs for iPSC and NPC marker genes.

(A) Violin plots of the cells in hiPSC-derived metanephric and mesonephric NPC populations reported in Tsujimoto et al. (2020) with standard quality control parameters after filtering. (B) UMAP plots for hiPSC-derived metanephric and mesonephric NPCs. The number of cells in each NPC population: metanephric NPCs, 190; and mesonephric NPCs, 199. (C, D) Violin plots of representative NPC (C) and iPSC (D) markers of hiPSC-derived metanephric and mesonephric NPCs. (E) UMAP plots of representative NPC markers (SIX2, PAX2, PAX8 and WT1) in hiPSC-derived metanephric and mesonephric NPCs. (F) Scatter plots of iPSC markers (LIN28A, CNMD and SFRP2) and NPC markers (SIX2 and PAX2). Numbers above the plots are Pearson correlation coefficients. Mesonephric NPCs were induced from hiPSCs by the same protocol as the metanephric NPC induction except for removing activin A at Stage 4. MESO, mesonephric NPC; META, metanephric NPC. (TIF)</p

Development of a digital droplet PCR assay for hiPSCs intermingled in NPCs derived from a clinical-grade iPSC stock line.

(A) Scatter plots of positive and negative droplets for digital droplet PCR (ddPCR) in an hiPSC/NPC cDNA dilution series sample. The purple lines indicate the cutoff values (left panels). A ddPCR analysis of MIR302CHG using an hiPSC cDNA dilution series sample in NPC cDNA shows a concentration-dependent change in the TBP-normalized copy number of MIR302CHG (right panel). (B) A representative result of ddPCR at annealing temperatures of 55°C to 65°C using 0.1% hiPSC/NPC cDNA samples shows a better separation of the positive and negative droplets of MIR302CHG and that TBP varies at an annealing temperature below 57.1°C. (C-E) Scatter plots of the ddPCR-estimated copy number ratios of TBP-normalized MIR302CHG using 5% (C), 15% (D) and 30% (E) of the RT products in the ddPCR reaction mix. *p p <0.01 by the Tukey-Kramer post hoc tests against the sample with 0% hiPSCs. NS, not significant.</p

Combination assay using magnetic bead-based cell isolation and qRT-PCR analysis to detect hiPSCs intermingled in NPCs derived from a clinical-grade iPSC stock line.

(A) Flow cytometric analysis of the MACS positive fractions of 1:1,000 mixtures of GFP (+) hiPSCs and GFP (-) NPCs at three different concentrations of anti-TRA-1-60 antibody-conjugated beads (upper panels) and one concentration of anti-SSEA-4 antibody-conjugated beads (lower left panel), GFP (-) NPCs (without MACS selection; lower center panel), and a 1:1,000 mixture of GFP (+) hiPSCs and GFP (-) NPCs (without MACS selection; lower right panel). The bar graph in the right panel shows the mean ± SEM of GFP (+) cells from the flow cytometric analysis at various bead concentrations in staining reagent. PF: positive fraction; NF: negative fraction; 0.2TRA-1-60: 0.2 μL TRA-1-60 beads / 1 μL staining reagent; 0.02TRA-1-60: 0.02 μL TRA-1-60 beads / 1 μL staining reagent; 0.002TRA-1-60: 0.002 μL TRA-1-60 beads / 1 μL staining reagent; 0.2SSEA-4: 0.2 μL SSEA-4 beads / 1 μL staining reagent. (B) Scatter plots of the TBP-normalized gene expressions of MIR302CHG, CUZD1 and POU5F1 in NPCs (NPC), the MACS negative fraction of the mixture (NF), 0.0001% hiPSC/NPC mixtures (10−4% iPSC), the MACS positive fraction of the mixture (PF), and hiPSCs (iPSC) by qRT-PCR analysis. (C) Scatter plots of the TBP-normalized gene expressions of MIR302CHG, CUZD1, and POU5F1 in day 4 cells (D4C), NF, 10−4% iPSC, PF and iPSC by qRT-PCR. The dots and lines in the center of the scatter plots in (B) and (C) indicate the experimental data and mean values of the data, respectively. UD: undetermined. *p p <0.01 by Tukey-Kramer post hoc tests against the sample with NPCs. NS, not significant.</p