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. 2019 Nov 11;8:e48767. doi: 10.7554/eLife.48767

Figure 5. 2F-hiSCs sustain the viability of mouse spermatogonia cells.

(A) Timeline and steps of co-culturing hiSCs with mouse germ cells. (B) Immunofluorescent staining of germ cell marker, DAZL (green), and human specific marker, NuMA (red), in co-cultured mouse spermatogonia cells with 2F-hiSCs or dH1-2K7 48 hr after plating. DAPI (blue) was used to indicate the nucleus. Scale bar = 50 μm. (C) Summary of cell numbers on the co-cultured plate in (B). Each data point indicates the number of cells counted in one separate image observed under 20 × fold microscope, area = 330 × 330 (μm). Error bars are the standard deviations of all the counted data points. dH1-2K7 (n = 18, technical replicates from two separated experiments), 2F-hiSCs (n = 25, technical replicates from two separated experiments). *p<0.05, **p<0.01, Student’s t-test. (D) Immunofluorescent staining of DAZL (green) and KRT18 (red) in co-cultured mouse spermatogonia cells and 2F-hiSCs 48 hr after plating. DAPI (blue) was used to indicate the nucleus. Scale bar = 50 μm. (E) Summary of cell numbers on the co-cultured plate in (D). Each data point indicates the number of cell counted in one separate image observed under 20 × fold microscope, area = 282 × 330 (μm). Error bars are the standard deviations of all the counted data points. dH1-2K7 (n = 4, technical replicates from two separated experiments), 2F-hiSCs (n = 9, technical replicates from two separated experiments). *p<0.05, **p<0.01, Student’s t-test.

Figure 5.

Figure 5—figure supplement 1. Isolation of mouse spermatogonia cells and co-culturing with hiSCs.

Figure 5—figure supplement 1.

(A–C) Bright field images of mouse seminiferous tubules after plating on culture plate. Arrow indicates location of spermatogonia cells attached to Sertoli cells. (D) Immunostainning of germ cell marker, DAZL, and Sertoli cell marker, SOX9, in cultured mouse seminiferous tubules. (E) Immunostainning of germ cell marker, DAZL and VASA, in cultured mouse seminiferous tubules. (F) Isolated spermatogonia cells immunostained with DAZL and VASA. (G) Isolated mouse spermatogonia cells plated on 2F-hiSCs or dH1-2K7. Bright field image was taken after 12 hr culturing. More round and healthy cells (arrow heads) attached to hiSCs than dH1-2K7 cells. Scale bar = 10 μm.
Figure 5—figure supplement 2. Immunofluorescent staining of germ cell marker, DAZL (green), and human specific marker, NuMA (red), in co-cultured mouse spermatogonia cells with aSCs or dH1-2K7 48 hr after plating.

Figure 5—figure supplement 2.

DAPI (blue) was used to indicate the nucleus. Scale bar = 50 μm. Summary of cell numbers on the co-cultured plates was shown on the left. Each data point indicates the number of cell counted in one separate image observed under 20 × fold microscope, area = 282 × 330 (μm). Error bars are the standard deviations of all the counted data points.