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

Abstract

Adult mammalian testis is a source of pluripotent stem cells. However, the lack of specific surface markers has hampered identification and tracking of the unrecognized subset of germ cells that gives rise to multipotent cells. Although embryonic-like cells can be derived from adult testis cultures after only several weeks in vitro, 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-beta-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.

Pluripotency of Male Germline Stem Cells

“…These cells exhibit a chimera formation efficiency of only 22%, express low levels of Rex1, Stra8, and Dazl, and express high levels of brachyury and Nog (Seandel et al, 2007). Earlier reports by Kanatsu-Shinohara et al (2004) and Guan et al (2006) clearly indicate that both ES-like and EpiSC-like colonies appear during derivation.…”

“…mGSs showed very similar morphology and gene expression patterns to mouse ES cells, and deve-loped into all 3 germ layers both in vitro and in vivo and contributed to chimera formation. Subsequent experiments showed that mGSs could also be derived from adult mice with various genetic backgrounds (Guan et al, 2006;Seandel et al, 2007). Clonal analysis in a separate experiment confirmed that they originated from SSCs (Ko et al, 2009).…”

“…In their model, cells respond to changes in the composition of the microenvironment and initiate fate conversion immediately, under proper in vitro culture conditions. In other studies, the reprogramming process is spontaneous but inefficient: reprogramming takes more than 4 weeks after derivation, depending on the experimental setup (Kanatsu-Shinohara et al, 2004;Seandel et al, 2007). Furthermore, frequent reprogramming events tend to occur in derivation steps with neonates (Kanatsu- Shinohara et al, 2004).…”

“…Culture conditions also affect reprogramming efficiency; the successful establishment of pluripotent stem cells is found exclusively in growth factor-rich Stempro34-based medium, which is optimized for hematopoietic stem cell culture and expansion, with irradiated MEFs as a feeder layer rather than under serum-free conditions on irradiated STO feeders. However, both methods are equally efficient for SSC maintenance (Kanatsu-Shinohara et al, 2004;Seandel et al, 2007).…”

“…Seandel et al (2007) successfully generated multipotent adult stem cells (MASCs) from GPR125 + spermatogonia using CD34 + irradiated mouse testicular stromal (CD34 + MTS) cells as feeders. Irradiated CD34 + MTS cells were effective both for the growth of GPR125 + spermatogonial progenitor cells as well as the emergence of MASCs.…”