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. 2021 Feb 19;4(5):e202000974. doi: 10.26508/lsa.202000974

Figure 7. Transcription factor-induced BT+AG+ cells differentiate into oogonia/gonocytes in xrOvaries.

Figure 7.

(A) A scheme for xrOvary culture (10, 11) with Dox-induced GATA3/SOX17/TFAP2C clone-derived d6 BT+AG+ cells. (B) Immunofluorescence analysis of TFAP2C (green), human mitochondria antigen (magenta), and DDX4 (cyan) expression (merged with DAPI) on aggregation day (ag) 77 xrOvaries. In two independent experiments, 28 and 23 TFAP2C/DDX4-expressing cells/7 sections, respectively, were detected. Bars, 50 μm. (C) The principal component analysis plots of the transcriptome of the GATA3/SOX17/TFAP2C clone-derived d6 BT+AG+ cells, d6ag77 BT+AG+ cells, and the parent clone-derived bone morphogenetic protein-induced d6ag77 BT+AG+ cells (see Table S1) with the relevant cell types during in vitro oogonia/gonocyte differentiation reported in reference 10, in which 585B1 BTAG hiPSCs (XY) and 1390G3 AGVT (AG; DDX4 [also known as VASA]-tdTomato [VT]) hiPSCs (XX) were used as starting materials. Numbers following “ag” indicate the culture days in xrOvaries. For the AGVT cells, ag77 and 120 AG+VT− (AG), AG+VT+ (AGVT) or AG−VT+ (VT) were used for analysis. (D) Expression dynamics of the key genes in GATA3/SOX17/TFAP2C clone-derived d6 BT+AG+ cells, and d6ag77 BT+AG+ cells (n = 2, red circles) (see Table S1), overlaid with those in the relevant cell types during the in vitro oogonia/gonocyte differentiation reported in reference 10. (E) Scatter-plot comparisons, combined with histogram representations (top and right of scatter plots), of the genome-wide 5 mC levels (genome-wide 2-kb windows) between the indicated cell types. (F) Violin-plot representation of the genome-wide 5 mC levels determined by whole-genome bisulfite sequence analysis in the cell types indicated. The mean levels are indicated by red bars. (G, H) Heat map representation showing the 5 mC levels in the indicated genomic elements on the autosomes (G) and in the differentially methylated regions of the indicated imprinted genes (H) in the indicated cells. HCP/ICP/LCP, high/intermediate/low-CpG promoters. The color coding is as indicated. (I) A model of the transcription factor circuitry driving human primordial germ-cell like cell specification.