Improved Efficiency of Female Germline Stem Cell Purification Using Fragilis-Based Magnetic Bead Sorting

Abstract

The enrichment of female germline stem cells (FGSCs) and the establishment of cell lines are influenced by the efficiency of cell purification. A previous study using mouse vasa homolog (MVH)-magnetic bead sorting for the isolation and purification of mouse FGSCs showed a relatively low efficiency. In this study, we tested 3 further proteins with the aim of improving the efficiency of FGSC purification. Immunofluorescence assays and magnetic sorting were performed using short-type pituitary gland and brain-cadherin (Stpb-c), CD9, and interferon-inducible transmembrane protein 3 (Iftm3, Fragilis), all of which are expressed in germ cells. Although all 3 proteins were expressed in FGSCs, CD9 was unsuitable because of its lack of germline specificity, and Stpb-c was also unsuitable because of the unavailability of an appropriate primary antibody. The efficiency of FGSC purification was remarkably enhanced using the germline-specific protein Fragilis, compared with that using MVH. This new method for the purification of FGSCs may have extensive applications in stem cell studies and clinical research.

Experimental evidence showing that no mitotically active female germline progenitors exist in postnatal mouse ovaries

“…These results showed that, under identical culture conditions as those reported by Zou et al (5, 7), the Ddx4-expressing cells from postnatal mouse ovaries did not enter mitosis during the 72-h cell culture. This is in contrast to the previous report that isolated FGSCs from the DDX-4 antibody-based purification started to proliferate within 24 h after seeding (7).…”

“…To evaluate the proliferation of Ddx4-expressing cells over a prolonged period, we cultured and passed the above-mentioned primary cells, as described by Zou et al (5,7). We found that the cultured testicular cells, as a positive control, formed RFP-positive cell colonies after 5-8 d of in vitro culture (Fig.…”

“…multifluorescent tracing | oogonial stem cells | female germline stem cells I t has been generally accepted for more than half a century that in most mammalian species oocytes cannot renew themselves in postnatal or adult life, and that the number of oocytes is already fixed in fetal or neonatal ovaries (1,2). This assumption, however, has been challenged over the past decade (3)(4)(5)(6)(7).…”

“…Recently, studies from primarily two research groups have put forward that functional female germline stem cells (FGSCs) and oogonial stem cells (OSCs) have been identified in adult mouse and human ovaries using antibody-based magnetic-activated cell sorting or fluorescence-activated cell sorting (4,5,7). Zou et al (5) reported that the FGSCs isolated from postnatal mouse ovaries were mitotically active during in vitro culture and that the FGSCs had begun to proliferate as early as 24 h after seeding (7).…”

“…Zou et al (5) reported that the FGSCs isolated from postnatal mouse ovaries were mitotically active during in vitro culture and that the FGSCs had begun to proliferate as early as 24 h after seeding (7). Moreover, these FGSCs were reported to be stable over 68 passages and to be able to differentiate into oocytes and to regenerate functional follicles when transplanted into the ovaries of recipient female mice that had been sterilized with chemotherapy drugs (5).…”