Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells

Kubota et al. 10.1073/pnas.0407063101.

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Fig. 5. Flow cytometric analyses of fresh and cultured magnetic-activated cell sorted (MACS) Thy-1 cells from C57xROSA pup testes. Fresh MACS Thy-1 cells were stained with antibodies against α v-integrin, α 6-integrin, and Thy-1 and analyzed by FACS (Upper). Live cell population (G1) was analyzed for α v-integrin expression (Upper Center). About 70% of G1 cells were α v-integrin^-/dim. The α v-integrin^-/dim cells (G2) were analyzed for α 6-integrin and Thy-1 expression (Upper Right). Cells in G2 were α 6-integrin⁺ Thy-1^lo/+. The fresh MACS Thy-1 cells were cultured on STO feeders with GDNF, soluble GFRα 1, and bFGF. After 2 weeks, the surface phenotype of the cultured cells was analyzed (Lower). The live cell population (G3) contained two distinct populations (Lower Center). One cell population (~75%), side scatter^hiα v-integrin⁺, represents primarily STO feeder cells. The second cell population (~25%) is side scatter^loα v-integrin^-/dim (G4), which is germ cell clump-forming cells. Cells in G4 express α 6-integrin and Thy-1 (Lower Right), an expression pattern similar to α v-integrin^-/dim cells of fresh MACS Thy-1 cells (Upper Right). The surface phenotype did not change during 6 months culture (data not shown).

Table 1. Stem cell activity and phenotypic analysis of cultured spermatogonial stem cells

*The number of spermatogenic colonies generated by 10⁵ cells transplanted into recipient testes (mean ± SEM).

^†Proportion of αv-integrin^-/dim α6-integrin⁺ Thy-1^lo/+ cells in cell suspension transplanted was determined by flow cytometric analysis.

^‡The number of spermatogenic colonies generated by 10⁵ αv-integrin^-/dim α6-integrin⁺ Thy-1^lo/+ cells transplanted into recipient testes (mean ± SEM).

Fig. 6. Flow cytometric analyses for gp130 and c-Kit expression on clump-forming germ cells. STO feeder cells were gated out, and the α v-integrin^-/dim germ cell population was analyzed (see Fig. 5). Filled histogram represents stained cells with the antibodies indicated. Open histogram indicates isotype control antibody-stained cells. Cultured spermatogonial stem cells (SSCs) expressed gp130. Only very weak expression of c-Kit was observed.

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