Supplementary Materials Supplemental Data supp_292_39_16003__index. sFGSCs, and E-cadherin converged in cellCcell connection regions, resulting Cholestyramine in the string-forming morphology. Our new method provides a platform for isolating FGSCs from the neonatal ovary, and our findings indicate that FGCSs exhibit string-forming features in neonatal mice. The sFGSCs represent a valuable resource for analysis of ovary function and an model for future clinical use to address ovarian dysfunction. for months, and viable offspring was obtained through transplantation of GFP-expressing FGSCs in ovaries (11). Human FGSCs were also isolated from reproductive-age women through DDX4 antibody-based FACS (12). GFP-expressing human FGSCs were injected into adult ovarian cortical tissue biopsies of humans, and the Cholestyramine ovarian tissue grafts were then xenografted into NOD-SCID female mice. GFP-positive oocytes can be detected in the tissue grafts, indicating their differentiation into oocytes (12). In addition to mice and humans, FGSCs from neonatal rats were also isolated by MACS and characterized (10). The rat FGSCs exert similar features of mice cells in both proliferation and differentiation. In addition, the neonatal FGSCs of both mice and rats were successfully used to generate transgenic or gene knockdown animals (10, 11, 18). Stably proliferating FGSCs can convert into female embryonic stemClike cells using embryonic stem cell medium, which exhibited gene expression and differentiation potential similar to those of embryonic stem cells (19). Comparison of gene expression profiles among FGSCs, primordial germ cells (PGCs), and SSCs revealed a similar pattern, but with distinct gene sets especially in stem cell markers (20, 21). Lineage-specific enhancers with germline stem cell features were also detected through comparison between embryonic stem cells (ESCs) and FGSCs. Their DNA methylation determined FGSC unipotency by suppressing the Rabbit Polyclonal to Synaptotagmin (phospho-Thr202) somatic program (9). Although some FGSCs or SSCs revealed a stringing growth pattern (21), the characterization of Cholestyramine the stringing growth or sFGSCs remains to be further studied. Antibody against the C terminus of Mvh (known as Ddx4 in humans) was first used for mouse FGSC isolation through MACS (11). In the subsequent studies, antibody against Fragilis (known as Ifitm3, a membrane protein), was used to isolate FGSCs from mice and rats through MACS (10, 13). Coupled with Mvh antibody, the FACS method was used for FGSC isolation from humans and mice (12). A FACS method was also used to isolate Oct4+ ovarian germline stem cells from Oct4-GFP transgenic mice (14). These isolation methods employed slightly different features of the cells; thus, the FGSCs isolated revealed distinct characteristics. Differential adherence selection was successfully used to enrich SSCs from postnatal testis (22,C24). As there was looser adherence of male germline stem cells compared with other somatic cells during culture (23, 24), we adopted the strategy of differential adherence selection to enrich female germ stem cells from the neonatal ovary. After 2-step digestions by collagenase IV and trypsin, dispersed ovary cells were selected by multiple rounds of differential adherence selections. Final detached cells were cultured for 3C5 passages, and the FGSCs were further characterized. We found the stringing FGSCs (sFGSCs) from primary to more than eight generations of culture. In addition, we tested mitotic kinetics and cell string-forming abilities of cultured sFGSCs. Membrane connection through E-cadherin and F-actin cytoskeleton of the cell Cholestyramine cortex in sFGSCs was also analyzed, which revealed tight connections between cells in Cholestyramine the sFGSCs. Our work demonstrated that sFGSCs exist in neonatal ovary, especially in 1C3-day postpartum (dpp) mice. Besides providing an alternative strategy for sFGSC isolation, which is much easier and costs less than FACS and MACS, the sFGSCs are valuable cell sources for further analysis of ovary functions and models for future clinic use of treating ovarian dysfunction. Results A methodological system of stringing FGSC isolation from neonatal ovaries through differential adherence selection In previous studies of ovary germline stem cells in mice and humans, antibodies against Mvh and Fragilis were used to isolate the stem cells through MACS (11, 13) and FACS (12). We adopted differential adherence selection to.