Rare stem cell population could enable new approaches to fertility treatment.

Scientists have isolated and purified rare, human germline stem cells that can be propagated over long periods of time in vitro and differentiate into follicles containing oocytes when transplanted into female mice. The achievement could pave the way to new therapeutic approaches in the field of human fertility, according to researchers at the Vincent Center for Reproductive Biology-led team at Massachusetts General Hospital, which led the work.

Such stem cells will also provide a more physiologically relevant in vitro model for studying human female germ cell development than embryonic stem cell-derived or induced pluripotent stem cell-derived germline cells currently used as models of human female gametogenesis. Jonathan L Tilly, M.D., and colleagues at the Vincent Center, Harvard Medical School, and Saitama Medical University in Japan, describe their findings in Nature Medicine in a paper titled “Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women.”

Evidence suggesting that female mouse ovaries harbor a rare population of germline egg-producing (oogonial) stem cells (OSCs) was first published by Dr. Tilly’s team in 2004. This controversial work essentially overturned what until then had been the fundamental belief that female mammals are born with a finite supply of eggs that are gradually used up throughout life. Subsequent work by teams around the world provided additional evidence to support the presence of OSC populations, in mice at least. This increasing evidence was topped in 2009 and 2010 by two publications demonstrating that OSCs isolated from neonatal and adult mouse ovaries could stably proliferate in vitro for months, spontaneously generate what appeared to be immature oocytes in culture and, when transplanted into adult mice, differ­entiate into mature eggs that were ovulated, fertilized, and produced viable offspring.

Despite this conclusive evidence in mice, there was still a lack of definitive evidence that ovaries of reproductive-age women contain a comparable population of oocyte-producing germline cells. Moreover, Dr. Tilly explains, even in mice it still wasn’t clear whether this adult oocyte pool could actually be renewed.

The Vincent Center researchers’ latest work was thus ultimately designed to develop a protocol for isolating, purifying, and maintaining human OSCs from human ovarian tissue and evaluating their differentiation potential in vitro and in vivo in mice recipients. The team based their studies on a recently reported protocol for obtaining OSCs from adult mouse ovaries, which identified and isolated the cell population by the using an antibody to a DEAD box poplypeptide 4 (Ddx4) variant, followed by immunomagnetic sorting.

The team adapted this protocol and, using an antibody directed against the C terminus of Ddx4, validated a fluorescence-activated cell sorting (FACS)-based approach to isolating OCS from ovaries of adult mice. By carrying out the technique on mouse ovaries from young animals, they estimated that about 0.014 +/- 0.002% of the total ovarian cell pool in mice is made up of OSCs.

The team then carried out the same technique on mouse and human ovarian tissue in parallel, to see whether DDX4-positive OCS could be collected from ovarian cortical tissue taken from healthy reproductive-age women. Encouragingly, freshly isolated Ddx4- and DDX4-positive cells from mouse and human ovaries were of similar size and morphology and exhibited matched gene-expression profiles that were rich in markers for early germ cells. Crucially, the authors add, and unlike Pou5f1-positive mouse embryonic stem cells, the mouse Ddx4-positive cells didn’t develop teratomas when transplanted into NOD-SCID mice. “Thus, although OSCs express numerous stem cell and primitive germ cell markers, these cells are distinct from the other types of pluripotent stem cells that have previously been described,” they write.

Notably, when actively dividing GFP-tagged mouse OSCs cultured in vitro were transplanted into the ovaries of nonchemotherapy-conditioned two-month old wild-type mice, GFP-expressing oocytes were detected in the follicles of animals stimulated to ovulate some 5–6 months later. After oviduct flushing, the team collected complexes comprising expanded cumulus cells surrounding oocytes both GFP+ and GFP-. Mixing these complexes with sperm from wild-type male mice resulted in fertilization and the development of preimplantation embryos, with the GFP-expressing fertilized eggs retaining GFP expression through the hatching blastocyst stage.

Both the mouse ovary-derived Ddx4-positive cells in vitro actively divided.  The rate of mouse OSC proliferation was approximately two­fold to threefold higher than that of the human OSCs maintained in parallel. A gene-expression analysis of the cultured cells also confirmed maintenance of early germline markers including several oocyte-specific markers.

Both the mouse OSCs and human DDX4-positive cells maintained in vitro generated spontaneous oocytes, and expressed known oocyte markers and, respectively, the mouse and human diploten-stage oocyte-specific marker Ybx2/YBX2, which is required for meiotic progression and gametogenesis. Having detected the meiotic marker YBX2 in the human OSC-derived oocytes, the researchers subsequently identified the presence of meiosis-specific proteins, while chromosomal DNA content analysis of the human OSC cultures revealed the presence of 4n and 1n cells, within the 2n population.    

Two sets of experiments supported the hypothesis that the in vitro-maintained human DDX4-positive cells were capable of generating viable oocytes and embryos. In vitro assays demonstrated that GFP-expressing DDX4-positive cells reaggregated into dispersed adult human ovarian cortical tissue and cultured generated large, GFP-positive cells enclosed within a structure of smaller GFP-negative cells resembling follicles. “We interpreted these findings as evidence that GFP-expressing human OSCs spontaneously generated oocytes that then became enclosed by somatic granulosa cells that were present in the adult human ovarian dispersates,” the authors write.

GFP-expressing human OSCs were then injected into human adult ovarian cortical tissue biopsies and xengrafted into NOD-SCID female mice. Fourteen days later, the grafts contained a number of immature follicles containing GFP-positive oocytes as well as the primordial and primary follicles containing GFP-negative oocytes, which would have been naturally present in the human tissue.

For technical, ethical, and legal reasons the researchers weren’t able to test whether the human DDX-expressing cells could generate viable embryos. Nonetheless, they state, “we have established a consistent and close parallelism between human ovary-derived DDX4-positive cells and mouse OSCs in terms of strategy of purification, yield from adult ovary tissue, morphology, primitive germline gene expression profile, in vitro growth properties, mitotic activity, meiotic activity, and the ability to form oocytes in defined cultures in vitro and in injected ovary tissue in vivo.

“We feel it is reasonable to conclude that the rare cells with cell-surface expression of DDX4 that are present in the ovaries of reproductive-age women represent adult human OSCs,” the authors conclude. “In addition to opening a new research field in human reproductive biology that was inconceivable less than 10 years ago, clear evidence for the existence of these cells in women may offer new opportunities to expand on and enhance current fertility-preservation strategies.”

The team says their experimental success was made possible at least in part due to three years of work spent refining the antibody-based protocol and the use of FACS, rather than immunomagnetic sorting. Their final technique allowed the Vincent team to confirm that while Ddx4 is located primarily inside oocytes, it is also present on the surface of OSCs.

The human ovarian cortical tissue samples provided by colleagues at Saitama, were obtained, with consent, from young healthy female subjects undergoing sex reassignment. The tissue had been vitrified, cryopreserved, and thawed prior to study, effectively demonstrating that the human OSCs can survival such manipulation with minimal loss of function.

“With assisted reproductive technologies involving cryopreservation of ovar­ian cortical tissue already in development for females with cancer, isolation and expansion of OSCs from this tissue before or after cryo­preservation might be useful for new fertility applications,” the team remarks. Moreover, and in contrast with current ESC- and iPSC-derived germline cells, “the availability of a detailed protocol for the purification of these newly discovered cells from human ovary tissue provides us and others with a much more physiologically relevant in-vitro model system from which to study human female germ cell development.”

Dr. Tilly’s team is currently exploring the establishment human OSC banks as, unlike oocytes themselves, OSCs can be frozen and thawed without damage. The researchers are also working to identify hormonal and other cues that promote the formation of oocytes from human OSCs and the potential develoment of OSC-derived mature human oocytes for IVF. 

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