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Supplementary information
Figure S1. Immunocytochemistry of germ cell markers on precursor cultures and PGCLCs.
A Immunofluorescence analysis for OCT4 (red), BLIMP1 (green), and STELLA (cyan) on days
1 and 2 of germ cell–precursor induction. Nuclei were stained with Hoechst (blue). Scale bar: 20
μm.
B Immunofluorescence analysis for OCT4 (green) and BLIMP1 (red) on day 5 of germ cell–
precursor induction. Nuclei were stained with Hoechst (blue). Scale bar: 20 μm.
C Immunofluorescence analysis for OCT4 (green) and cKIT (red) on day 7 of germ cell–
precursor induction. Nuclei were stained with Hoechst (blue). Scale bar: 20 μm.
Figure S2. Induction of TRA-1-81+/cKIT+ PGC-like cells.
A Effect of SCF during PGC-like cell induction on PGC-like cell survival of d6 cultures.
B Induction of PGC-like cells from HuES6 ESCs and SA8 iPSCs on day 4.
Figure S3. FACS analysis of cKIT+/TRA-1-81+ and cKIT+/SSEA1+ PGCLCs on day 4.
A Isotype control for cKIT/Tra-1-81 gating (left panel); cKIT+/TRA-1-81+ sorting (right panel).
B Isotype control for cKIT+/SSEA1+ gating (left panel); cKIT+/SSEA1+ sorting (right panel).
Figure S4. Effects of KSR and WNT3A on the expression of selected trophecto-, ecto-, endo, and mesodermal genes during PGC-precursor induction.
qRT-PCR analysis of d2 precursor cells that were induced in increasing KSR concentrations in
the presence or absence of WNT3A (100 ng/ml). Samples were calibrated with iPSC values, and
iPSC values depict 1. Y-axes are in log2 scale.
Figure S5. Expression of core PGC genes.
A Heatmap of the transcriptomics patterns of the core PGC genes. The colorbars codify the gene
expression in log2 scale. Red corresponds to high gene expression.
B Violin plots of the expression distribution of the core PGC genes. The red squares represent the
position of the means. The blue points represent the spread of the expression of the genes used to
build the distributions.
Figure S6. Expression of mesoderm markers.
A Heatmaps. Heatmap of the transcriptomics patterns of the mesoderm markers. The colorbars
codify the gene expression in log2 scale. Red corresponds to high gene expression. B Violin plots
of the expression distribution of the mesoderm markers. The red squares represent the position of
the means. The blue points represent the spread of the expression of the genes used to build the
distributions.
B Violin plot associated with the expression distribution of the mesoderm markers. The red
squares represent the position of the means. The blue points represent the spread of the
expression of the genes used to build the distributions.
Figure S7. Expression of SOX family members
A Heatmap of the transcriptomics patterns of the SOX-family members represented in the
Illumina MouseRef-8 v2 transcriptomics microarrays. The colorbars codify the gene expression
in log2 scale. Red corresponds to high gene expression.
B Violin plots of the expression distribution of SOX-family genes. The red squares represent the
position of the means. The blue points represent the spread of the expression of the genes used to
build the distributions.
Figure S8. Expression of PRDM14-regulated-genes.
A Heatmap of the transcriptomics patterns of the PRDM14-regulated genes. The colorbars
codify the gene expression in log2 scale. Red corresponds to high gene expression.
B Violin plots of the expression distribution of the PRDM14-regulated genes. The red squares
represent the position of the means. The blue points represent the spread of the expression of the
genes used to build the distributions.
Figure S9. Plot bar of the -log10(p) of the significant enriched GO terms of down-regulated
(A) and up-regulated (B) human d6 PGCLCs is relation to iPSCs.
The longer the bar, the higher is the statistical significance of the enrichment (p-values are
written within parentheses).
Figure S10. Effect of knockdown of BLIMP1 and PRDM14 on d4 cKIT+/TRA-1-81+
PGCLCs induction.
The induction of d4 PGCLCs was analyzed by FACS, gated for TRA-1-81 and cKIT. A
representative FACS plot image of d6 PGCLCs from each cell population infected by control and
shBLIMP1 and shPRDM14 lentivirus is shown.
Figure S11. Immunocytochemistry for PRDM14 on precursor cultures.
Immunofluorescence analysis for PRDM14 (red) on days 0, 1, and 2 of germ cell–precursor
induction. Nuclei were stained with Hoechst (blue). Scale bar: 100 μm.
Table S1 List of primers used in this study.