FIGURE 2. Characterization and multipotent derivatives of Gpr125^lacZ/lacZ SPC lines

a, Morphology of SPC colonies and expression of GPR125 by X-gal staining (blue, inset). b, Proliferation of GPR125⁺ SPCs (GSPCs) in culture. c, Immunolabelling by germ-cell markers GCNA (brown, left panel), and anti-DAZL (brown, right panel). Absent staining in feeders is denoted by asterisks. d, Expression of GPR125–LacZ in cloned GSPCs (tracked by GFP (green, inset), using lentivirus). e, Quantitative PCR of Gpr125^lacZ/lacZ GSPCs compared to Gpr125^lacZ/lacZ total testis. Bars depict fold change ( scaled s.d.) of gene expression (compared with total testis) of genes associated with GSPCs or differentiating spermatogenic cells. f–h, Engraftment of Gpr125^lacZ/lacZ GSPCs microinjected into busulphan-treated testes. f, Confocal slices (1 m, inset) distinguishing areas with GFP^bright spermatogonia along the basement membrane (arrows) from centrally located areas containing smaller, round GFP^dim differentiating cells, in the projection of 32 slices. g, GPR125 expression by X-gal staining (blue, arrowheads) present in engrafted cells along the basement membrane. h, Differentiation of donor-derived GFP⁺ cells and GFP^- non-engrafted tubules (arrowheads, GFP⁺ spermatids; asterisk, non-engrafted tubule). i, Derivation of GPR125⁺ MASCs (blue staining in inset, X-gal) from GSPCs. j, Nuclear labelling by anti-OCT4 (brown). k, Flow cytometry for GPR125 expression in Gpr125^lacZ/lacZ MASCs or GSPCs by FDG staining (mean fluorescence intensity: 22.1 or 18.2, respectively, versus 2.2 in wild-type GSPC control). Scale bars, 50 m.