Human female germline stem cells (FGSCs) have opened new opportunities for understanding human oogenesis, delaying menopause, treating infertility, and providing a new strategy for preserving fertility. However, the shortage of adult human ovaries tissues available impedes their future investigations and clinical applications. Here, we have established FGSC lines from scarce ovarian cortical tissues that exist in follicular aspirates (faFGSCs), which are produced and discarded in in vitro fertilization centers worldwide. The faFGSCs have characteristics of germline stem cells involved in the gene expression profile, growth characteristics, and a normal karyotype consistent with that of FGSCs obtained from ovarian cortexes surgically removed from patients (srFGSCs). Furthermore, faFGSCs have developmental potentials including spontaneous differentiation into oocytes under feeder-free conditions, communicating with granulosa cells by gap junctions and paracrine factors, entering meiosis after RA induction, as well as forming follicles after injection into human ovarian cortical tissues xenografted into adult immunodeficient female mice. Lastly, we developed a strategy guiding FGSCs differentiated into germinal vesicle (GV) stage oocytes in vitro and revealed their developmental mechanisms. Our study not only provides a new approach to obtain human FGSCs for medical treatment, but also opens several avenues to investigate human oogenesis in vitro.

Pigs share many anatomical and physiological features with humans, offering a unique and viable model for biomedical research. Although porcine female germline stem cells (FGSCs) were identified in the juvenile ovary, no reports described the isolation and purification of FGSCs from the pig at sexual maturity. Here, we isolated, purified, and cultured FGSCs from porcine ovaries at sexual maturity. Furthermore, we established and characterized the porcine FGSC (pFGSC) lines. In addition, we found that pFGSC lines could differentiate into oocytes when injection into tissue grafts, including human ovarian tissues. The results show that FGSCs exist in ovaries of Banna mini-pigs at juvenile and sexually maturity. These findings have implications in animal biotechnology applications and regeneration medicine.