A) Schematic representation of piggyBac over expression constructs. Transposase expression vector (piggyBac) contained the IFP2 open reading frame (ORF) driven by the CAG promoter. Transposon expression vectors contained the ORF for NANOS3 (pbNANOS3) or DAZL (pbDAZL) driven by the CAG promoter, followed by IRES element and puromycin resistance gene, and flanked by the piggyBac 5’ and 3’ long terminal repeats (LTRs). Vector without ORF was used as a control (pbMOCK). Human ES cells were co-transfected with the transposase vector and one of the transposon vectors, and selected with puromycin. B) Gene expression analysis of NANOS3 and DAZL expression by qPCR. Samples from three separate transfections were used at passage 2 after transfection. Values are relative quantities normalized to GAPDH and RPLPO, and represented as mean ± SD. Statistical significance was tested by one-way ANOVA, **** p < 0.0001, *** p < 0.001. See also . C) Immunofluorescence staining of NANOS3 (red) and DAZL (red) in pbNANOS3 and pbDAZL cells, respectively. Note the DAZL positive cells outside the colonies. pbMOCK cells were used as negative control. Cells were counterstained with DAPI (blue). Scale bar 100 μm. D) Confirmation of NANOS3 and DAZL protein expression in pbNANOS3 and pbDAZL cells by Western blotting. Samples at passage 2, 5 and 6 after transfection were used for each cell line. pbMOCK cells were used as negative control, and GAPDH was used as endogenous control for protein loading. See also . E) Gene expression of pluripotency markers OCT4 and NANOG in transfected cells. Samples from three separate transfections were used at passage 2 after transfection. Values are relative quantities normalized to GAPDH and RPLPO, and represented as mean ± SD. See also . F) Immunofluorescence staining of OCT4 (green) and NANOG (red). Note the negative pbDAZL cells outside the colonies. Cells were counterstained with DAPI (blue). Scale bar: 100 μm. See also .

A) Differential gene expression analysis for transfected cells by mRNA sequencing. Samples from three separate transfections were used at passage 2 after transfection. Results are shown in MA-plot with log fold change (logFC) values in the pbNANOS3 or pbDAZL versus the pbMOCK sample, plotted against average log counts per million (logCPM). Significant genes (FDR < 0.05) are marked as red: pbNANOS3 (4 genes), pbDAZL (38 genes). See also . Selected differentially expressed genes for B) pbNANOS3 and C) pbDAZL, with suggested functions. Values are Fragments Per Kilobase Of Exon Per Million Fragments (FPKM), and represented as mean ± SD. See also and Figs.

A) Gene expression analysis for cells differentiated on SNL-feeders using SSC medium for 5, 7, 10 or 14 days, and for cells in undifferentiated culture conditions (D0). Samples from three separate experiments were used at passage 6–8 after transfection. Values are relative quantities normalized to GAPDH and RPLPO, and represented as mean ± SD. Statistical significance was tested by two-way ANOVA, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05. See also . B) Immunofluorescence staining of transfected cells differentiated for 14 days on SNL-feeders; PLZF (green), GFRa1 (red), NANOS3 (red) and DAZL (red). Cells were counterstained with DAPI (blue). Scale bars: 100 μm. See also .

A) Weight of the xenografts collected 8 weeks after transplantation, n ≥ 3. B) Whole mount staining with an antibody against primate testis cells. A cell clump was observed in one pbNANOS3 transplanted testis. Six colonies with spermatogonial characteristics were observed in one pbDAZL transplanted testis, representative image shown. Scale bar: 100 μm. See also . C) Serial sections from xenografts with weight < 150mg were screened for intratubular NuMA positive cells and percentage of positive tubules was calculated. Data are represented as mean ± SD, n ≥ 3. See also . D) Immunofluorescence staining for human specific NuMA (red) and DDX4 (green). Human fetal testis sample was used as positive control. Representative images with intratubular NuMA positive and DDX4 negative cells are shown for pbMOCK, pbNANOS3 and pbDAZL xenografts. Cells were counterstained with DAPI (blue). Scale bar: 100 μm.