Fig 1: Gene expression properties are maintained in SSCs cultured with KSR.(A) Relative expression levels of marker genes for undifferentiated spermatogonia (Plzf) and ESCs (Nanog). Data are presented as means ± s.d. from three independent experiments. The Y-axis indicates the relative expression level, with the value of MEF_B10K set to 1 for each group. (B) Relative expression levels of other undifferentiated spermatogonial markers (Gfra1, Etv5, Lhx1, Bcl6b, and Neurog3) and differentiated spermatogonial markers (c-Kit and Stra8). Data are presented as means ± s.d. from three independent experiments. The Y-axis indicates the relative expression level, with the value of MEF_B10K set to 1 for each group. (C) Cell cycle analyses by flow cytometry at 5 days after the medium change from MEF_B10K to each indicated condition. Histograms are representative examples of each sample. The tables under each histogram indicate the percentages of each cell cycle stage. The histograms are colored according to each cell cycle stage. Green: G1 phase. Ocher: S phase. Light blue: G2/M phase. (D) Quantification of the results shown in (C). The graph is presented as means of the percentages ± s.d. from three independent experiments. Asterisks indicate statistical significance (*p<0.05, **p<0.01, ANOVA, Tukey’s HSD test).
Fig 2: GDNF is indispensable but can be reduced in the presence of KSR.(A) Growth curves of SSCs under reduced cytokine conditions (left panel), summarized in the table (right panel). The number of SSCs was counted at the indicated weeks, and the cell counts are presented as means ± s.d. from three independent experiments. No proliferation was observed in Null condition. The calculated doubling times are also shown in the table (right panel). N.D., not determined due to inefficient proliferation. (B) Relative mRNA expression levels of undifferentiated spermatogonial markers (Gfra1, Etv5and Neurog3) and differentiating/differentiated spermatogonial markers (c-Kit and Stra8) were examined at 12 hours after the medium change from the full cytokine conditions to the indicated conditions. RA48 cells are differentiated SSCs treated with retinoic acid for 48 hours. Data are presented as means ± s.d. from three independent experiments. The Y-axis indicates relative expression level to the value of Full, set to 1 for each group.
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