Additional file 1 of Human primordial germ cell heterogeneity in vitro is associated with distinctive biological states
Year: 2026
Type: article
Abstract: Additional file 1. Figure S1. scRNA-seq of hPGCLC extended culture. UMAP plot of scRNA-seq from two replicates of D4C21 Feature plots for representative gene expression from D4C21. Top; transcription factors for pluripotency, bottom; post-migratory PGC markers. Dot plot presenting functional terms identified for the cluster 1and 2. Figure S2. Generation and validation of TFAP2C-eGFP and DND1-tdTomato dual reporter hESC line. Schematic representation of CRISPR-Cas9 targeting strategy for TFAP2Cand DND1loci. The constructs for knocking-in 2A-eGFP into the TFAP2C or 2A-tdTomato into the DND1 loci are also shown. Black boxes indicate the exons. Arrows indicate PCR screening primers. Genotyping PCR analysis of targeted clones for TFAP2C, DND1, and random integration of the targeting vector. Rec., recombined by Cre; non, non-targeted. Yellow arrow, 4 kb; yellow asterisk, 1.5 kb. Clone 16was selected for the AGDT hESC line Representative karyotype for AGDT showing normal 46, XY chromosomal complement. Phase-contrast image of AGDT hESC line. Phase-contrast image of AGDT-derived iMeLCs. Phase-contrastand epi-fluorescenceimages of hPGCLCs derived from AGDT hESC for TFAP2C-eGFP Phase-contrast imagesand flow cytometry analysisof xrTestes aggregates at d2 and ALI culture at d2 + 21 . Phase-contrastand TFAP2C-eGFP epi-fluorescenceimages of extended culture at c7 and c14. Yellow dashed lines outline TFAP2C-eGFP + hPGCLC colonies. Flow cytometry analysis of TFAP2C-eGFP and DND1-tdTomato expression during extended culture at c12, c22, and c35 without enrichment by tdTomato. Scale bars in E-I, 200 μm. Scale bar in J, 50 μm. Figure S3. Immunostaining and cell cycle analysis in extended culture at c42. IF images for P21with TFAP2Cand RFPin DND1 + at c42. IF images for P21and P27with TFAP2C-eGFPin DND1- hPGCLCs at c42. Scale bars = 20 µm and nuclei are stained with DAPI. Cell cycle analysis of DND1- and DND1 + hPGCLCs by EdU and DAPI corporation at c42.EdU and DAPI analysis of DND1 and DND1 + hPGCLCs by FACS. The average percentage of cells in the indicated cell cycle stages are shown.Bar graph of the percentage of each cell cycle stage. Error bars, SD. Figure S4. Comprehensive transcriptomic comparisons during extended culture. 2D PCA plots showing pairwise comparisons of transcriptomes. Left: PC1 vs PC2; Middle: PC1 vs PC3; Right: PC2 vs PC3. Black circles, d5_hPGCLC; cyan circles, c42_DND1 −; yellow circles, c42_DND1 +. Volcano plots comparing gene expression between d5_hPGCLC and extended culture populations. Left: d5_hPGCLC vs c42_DND1 −; Right: d5_hPGCLC vs c42_DND1 +. Red dots indicate up-regulated genes; blue dots indicate down-regulated genes. Numbers of differentially expressed genes are shown. Heatmap showing expression patterns of all differentially expressed genes unique to c42_DND1 − and c42_DND1 + hPGCLCs, compared to hPGCLC at d5. Hierarchical clustering reveals distinct transcriptional programs. Heatmap of genes associated with male gamete generation across biological replicates of d5 and c42 DND1 −, and DND1 + hPGCLCs. Gene names are listed on the right. Color scale represents Log2. Figure S5. DNA methylation status in extended culture. Dot plot showing global CG methylation levels in hPGCLCs at d5, DND1-, DND1 + hPGCLCs at c42 and female hPGCs at week 5.5. Statistically significant differences were identified with One-way ANOVA. *p = 0.0057 between d5_hPGCLC and c42_DND1 +, *p < 0.0001 compared to hPGC Wk5.5 F. The DNA methylome data of hPGC is from SRP057098. The whole-genome bisulfite sequencing analysis covered 22.7 K CpG sites in d5_hPGCLC, 10.6 K CpG sites in c42_DND1 − , and 5.7 K CpG sites in c42_DND1 + . Violin plots for the distribution of overall CG methylation level. Average CG methylation levels over protein coding genes and 2 kb flanking regions TSS, transcription start site; TTS, transcription termination site. CpG methylation at gene bodies of differentially expressed gene sets. Figure S6. BMP2 supplement on hPGCLC extended culture. A FACS plot and a bar graph of hPGCLC cultured with BMP2 from c0-c14. The average percentage for each population is shown. Gray and black bars indicate the percentage of DND + and DND1- hPGCLCs, respectively. Error bars, SD. Gene expression analysis of DAZL and DDX4 at c42 in DND1 + cells cultured with and without BMP2. qPCR was conducted in triplicate and normalized to expression of the average of ACTB and GAPDH. Error bars, SEM. Statistically significant differences were identified with a two-tailed t-test. ns, non-significance.
Source: Figshare
Institution: University of California, Los Angeles
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7212188675