Gametogenesis in a dish

npg Cell Research (2012) 22:1422-1425. © 2012 IBCB, SIBS, CAS All rights reserved 1001-0602/12 $ 32.00 www.nature.com/cr 1422 RESEARCH HIGHLIGHT Gametogenesis in a dish Ying Gu1, Guang-Hui Liu1, 2, Juan Carlos Izpisua Belmonte1, 3 Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA; 2National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; 3Center for Regenerative Medicine in Barcelona, Dr. Aiguader 88, 08003 Barcelona, Spain 1 Cell Research (2012) 22:1422-1425. doi:10.1038/cr.2012.84; published online 29 May 2012 Recent progress in the induced pluripotent stem cell (iPSC) field as well as the establishment of germline stem cell isolation and culture methodologies may provide an in vitro platform for the study of physiological and pathological human gamete development and open new avenues for cell replacement-based personalized treatment of infertility. Human infertility affects many couples worldwide. Based on a report from the World Health Organization (WHO), about 10%-15% of the reproductive aged population is infertile and around 25% of married women of reproductive age in developed countries suffer from infertility. Human infertility is frequently linked to defective gamete (oocyte or sperm) development, which can be triggered by genetic mutations or by environmental factors. In addition, high dose anti-cancer treatments also lead to gamete damage, and consequently leave patients with transient or permanent infertility. For patients with reduced oocyte or sperm quality, gamete donation, a process that is accompanied by a series of ethical, personal and legal concerns, is the only available option. Recent exciting achievements in gamete Correspondence: Juan Carlos Izpisua Belmontea, Guang-Hui Liub a E-mail: , b E-mail: in vitro formation from either gonadal stem cells or pluripotent stem cells, including embryonic stem cells (ESCs) and iPSCs, have not only broadened our knowledge of human gamete development and related disorders, but also may hold great promise for the development of alternative infertility treatments. In males, continual spermatogenesis during adult life is dependent on an adult germline stem cell population known as spermatogonial stem cells (SSCs). These cells reside at the base of seminiferous tubules of the testes, can be identified by their ability to generate a colony through spermatogenesis after transplantation into the testes of germ cell-deficient recipients [1], and can be isolated from testes and maintained and cultured in vitro [2]. Contrary to this proven experimental observation, the existence of female germline stem cells has remained controversial for many years. Indeed, it has been a longstanding belief that female mammals lose the capability of restoring their oocyte population after birth. This assumption was greatly challenged by Johnson et al. [3] in 2004, when they identified oogonial stem cells (OSCs) that sustain oocyte generation in both juvenile and adult mouse ovaries. Subsequent studies by Zou et al. [4] showed that these female germline stem cells could be isolated from adult mouse ovaries by immunomagnetic bead sorting and expanded long term in vitro. However, the iden- tification, isolation and in vitro culture conditions of human OSCs remained to be fully elucidated. Recently, White et al. [5] reported the isolation of OSCs from both mouse ovaries and human ovarian cortical tissue of reproductiveaged women. They used an antibody against the surface variant of the DEAD box polypeptide 4 (Ddx4), a germ cellspecific RNA helicase, to detect the putative OSCs in the dissociated ovarian tissue. Utilizing the externally exposed epitope of Ddx4, they purified the viable OSCs via a fluorescence-activated cell sorting (FACS)-based protocol. Compared to the previous study that used magnetic bead sorting to isolate mouse OSCs [4], this FACS-based protocol decreased the possibility of contamination with non-targeted cells and dead or damaged cells, and also allowed for the measurement of other cellular properties of the sorted OSCs. This study also demonstrated that the isolated OSCs are actively dividing and can be expanded for months in a culture dish. More importantly, they can spontaneously generate oocytes under appropriate in vitro and in vivo conditions. This work strongly supports earlier studies [3, 4], which indicated that the ovaries of female mammals maintain the ability to produce oocytes during adulthood. Altogether, these recent observations may have revolutionary implications for human health, including infertility treatments and fertility preservation Cell Research | Vol 22 No 10 | October 2012 npg npg 1423 Figure 1 Proposed model for in vitro gametogenesis from iPSCs or germline stem cells. The germline stem cells (SSCs from males, or OSC from females) could be isolated, cultured, and transplanted back to patients to restore fertility. Meanwhile, in vitro gametogenesis could be induced from both germline stem cells and patients-derived iPSCs to generate oocytes and sperm for both research and therapeutic purposes. Gene editing could be applied in cultured iPSCs, and germline stem cells to correct disease-related mutations. iPSCs: induced pluripotent stem cells. SSCs: spermatogonial stem cells. OSCs: oogonial stem cells. for female patients receiving cancer treatments. Animal models, especially the mouse, together with in vitro approaches, including research on ESCs and iPSCs, have and will provide the conceptual and practical knowledge necessary for these expectations to be realized. For instance, for patients with defects in early germ cell development, research on ESC and iPSCs opens a hopeful alternative for gamete regeneration. A number of labs have reported the in vitro derivation of germ cell lineages from murine ESCs [6-10]. For female gamete generation, Hubner et al. [6] differentiated mouse ESCs containing a GFP reporter under the control of a truncated, germ cell-specific www.cell-research.com | Cell Research promoter for Oct4 in an adherent culture system, and enriched the GFP-positive aggregates for further culture for several weeks. They subsequently observed the formation of multicellular structures resembling ovarian follicles, as well as the extrusion of oocyte-like cells from these structures. Although the oocytes generated in their culture system bear parthenogenesis activity, there was no evidence indicating that these oocytes could be fertilized. In contrast to the 2D culture method employed for derivation of female gametes, two other groups [7, 8] created male germ cells from mouse ESCs through 3-dimensional embryoid body (EB) culture. The male germ cells generated from the EB culture could either express spermatozoid markers when transplanted into the testis of male mice [7] or fertilize oocytes by intracytoplasmic injection [8], raising t (...truncated)