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Generation of functional multipotent adult stem cells from GPR125+ germline progenitors


Adult mammalian testis is a source of pluripotent stem cells1. However, the lack of specific surface markers has hampered identification and tracking of the unrecognized subset of germ cells that gives rise to multipotent cells2. Although embryonic-like cells can be derived from adult testis cultures after only several weeks in vitro1, it is not known whether adult self-renewing spermatogonia in long-term culture can generate such stem cells as well. Here, we show that highly proliferative adult spermatogonial progenitor cells (SPCs) can be efficiently obtained by cultivation on mitotically inactivated testicular feeders containing CD34+ stromal cells. SPCs exhibit testicular repopulating activity in vivo and maintain the ability in long-term culture to give rise to multipotent adult spermatogonial-derived stem cells (MASCs). Furthermore, both SPCs and MASCs express GPR125, an orphan adhesion-type G-protein-coupled receptor. In knock-in mice bearing a GPR125–β-galactosidase (LacZ) fusion protein under control of the native Gpr125 promoter (GPR125–LacZ), expression in the testis was detected exclusively in spermatogonia and not in differentiated germ cells. Primary GPR125–LacZ SPC lines retained GPR125 expression, underwent clonal expansion, maintained the phenotype of germline stem cells, and reconstituted spermatogenesis in busulphan-treated mice. Long-term cultures of GPR125+ SPCs (GSPCs) also converted into GPR125+ MASC colonies. GPR125+ MASCs generated derivatives of the three germ layers and contributed to chimaeric embryos, with concomitant downregulation of GPR125 during differentiation into GPR125- cells. MASCs also differentiated into contractile cardiac tissue in vitro and formed functional blood vessels in vivo. Molecular bookmarking by GPR125 in the adult mouse and, ultimately, in the human testis could enrich for a population of SPCs for derivation of GPR125+ MASCs, which may be employed for genetic manipulation, tissue regeneration and revascularization of ischaemic organs.

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Figure 1: Restricted GPR125 expression in adult mouse testis and derivation of multipotent cells from spermatogonial progenitor cells (SPCs).
Figure 2: Characterization and multipotent derivatives of Gpr125 lacZ/lacZ SPC lines
Figure 3: GPR125–LacZ MASCs exhibit multipotency and can form functional vessels.
Figure 4: Gpr125 lacZ/lacZ MASCs have an expression profile different from mouse embryonic stem cells.


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This work was supported by the Howard Hughes Medical Institute, Ansary Stem Cell Center for Regenerative Medicine and Memorial Sloan Kettering Cancer Center T32 grant (M.S.), an AACR–Genentech BioOncology Fellowship for Cancer Research on Angiogenesis (M.S.), the Heed Foundation (S.C.), the International Retinal Research Foundation (S.C.) and National Heart, Lung and Blood Institute grants (S.R.). We thank M. Hardy, P. Schlegel, Marc Goldstein, A. Brivanlou and S. Noggle for critical input. We are grateful to G. Enders for providing anti-GCNA antibody. We thank D. S. Johnston, G. Linkov and G. Zlotchenko for technical assistance.

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Correspondence to Shahin Rafii.

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S.R., M.S., S.V.S. and S.C. have filed a provisional patent application related to the use of GPR125.

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Seandel, M., James, D., Shmelkov, S. et al. Generation of functional multipotent adult stem cells from GPR125+ germline progenitors. Nature 449, 346–350 (2007).

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