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

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Abstract
Adult stem cells are an attractive alternative to embryonic stem cells for therapeutic use. As yet there is no standard method for obtaining such cells from adults and priming them to form different tissues, but a new system that generates large numbers of stem cells from the adult testicle shows promise. It makes use of a novel marker, an orphan receptor known as GPR125, found on the surface of spermatogonial stem cells. The use of specialized feeder cells to support stem cell growth allows stem cells once destined for spermatogenesis to become multipotent. This work also provides clues as to the minimal requirements for multipotency in adult cells. Adult stem cells are an ethically attractive alternative to embryonic stem cells for obtaining tissues for therapeutic purposes. However, no standardized methods exist to obtain such cells reliably from adults, and then convert them into different tissues. However, a system has been developed to generate large numbers of stem cells from the adult testicle, based on the presence of a novel marker (GPR125), specifically on the stem cell surface. 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.