Cell sorting technique isolates egg-producing stem cells from adult human ovaries

Researchers at Massachusetts General Hospital (MGH; Boston, MA)—using a fluorescence-activated cell sorting technique—have isolated egg-producing stem cells from the ovaries of reproductive-age women and shown that these cells can produce what appear to be normal egg cells (oocytes). The research could pave the way for a method to overcome infertility in women and perhaps delay the timing of ovarian failure, and follows up their 2004 Nature paper that first suggested female mammals continue producing egg cells into adulthood.

Back in 2009, in work published in Nature Cell Biology, a research team at Shanghai Jiao Tong University in China determined that oocyte-producing stem cells (OSCs) often result in the contamination of desired cells by other cell types. To address this, the team developed and validated a much more precise cell sorting technique to isolate OSCs without contamination from other cells. The study also had isolated OSCs based on cell-surface expression of a marker protein called Ddx4, which previously had been found only in the cytoplasm of oocytes. This apparent contradiction with earlier studies raised concerns over the validity of the protocol. So the MGH-Vincent team, using their fluorescence-activated cell sorting techniques, verified that, while the marker protein Ddx4 was indeed located inside oocytes, it was expressed on the surface of a rare and distinct population of ovarian cells identified by numerous genetic markers and functional tests as OSCs.

To examine the functional capabilities of the cells isolated with their new protocol, the MGH-Vincent team injected green fluorescent protein (GFP)-labeled mouse OSCs into the ovaries of normal adult mice. Several months later, examination of the recipient mouse ovaries revealed follicles containing oocytes with and without the marker protein. GFP-labeled and unlabeled oocytes also were found in cell clusters flushed from the animals' oviducts after induced ovulation. The GFP-labeled mouse eggs retrieved from the oviducts were successfully fertilized in vitro and produced embryos that progressed to the hatching blastocyst stage, a sign of normal developmental potential. Additionally, although the Chinese team had transplanted OSCs into ovaries of mice previously treated with chemotherapy, the MGH-Vincent team showed that it was not necessary to damage the recipient mouse ovaries with toxic drugs before introducing OSCs.

In their last two experiments, the MGH-Vincent team used their new cell sorting techniques to isolate potential OSCs from adult human ovaries. The cells obtained shared all of the genetic and growth properties of the equivalent cells isolated from adult mouse ovaries, and like mouse OSCs, were able to spontaneously form cells with characteristic features of oocytes. Not only did these oocytes formed in culture dishes have the physical appearance and gene expression patterns of oocytes seen in human ovaries—as was the case in parallel mouse experiments—but some of these in vitro-formed cells had only half of the genetic material normally found in all other cells of the body. That observation indicates that these oocytes had progressed through meiosis, a cell-division process unique to the formation of mature eggs and sperm.

The researchers next injected GFP-labeled human OSCs into biopsied human ovarian tissue that was then grafted beneath the skin of immune-system-deficient mice. Examination of the human tissue grafts 7 to 14 days later revealed immature human follicles with GFP-negative oocytes, probably present in the human tissue before OSC injection and grafting, as well as numerous immature human follicles with GFP-positive oocytes that would have originated from the injected human OSCs.

"These experiments provide pivotal proof-of-concept that human OSCs reintroduced into adult human ovarian tissue performed their expected function of generating new oocytes that become enclosed by host cells to form new follicles," says Tilly. "These outcomes are exactly what we see if we perform the same experiments using GFP-expressing mouse OSCs, and GFP-expressing mouse oocytes formed that way go on to develop into fully functional eggs."

"In this paper we provide the three key pieces of evidence requested by those who have been skeptical of our previous work," adds Tilly. "We developed and extensively validated a cell-sorting protocol to reliably purify OSCs from adult mammalian ovaries, proving once again that these very special cells exist. We tested the function of mouse oocytes produced by these OSCs and showed that they can be fertilized to produce healthy embryos. And we identified and characterized an equivalent population of oocyte-producing stem cells isolated from adult human ovaries."

Potential clinical applications for these findings include the establishment of human OSC banks—since these cells, unlike human oocytes, can be frozen and thawed without damage—the identification of hormones and factors that accelerate the formation of oocytes from human OSCs, the development of mature human oocytes from OSCs for in vitro fertilization (IVF), and other approaches to improve the outcomes of IVF and other infertility treatments.

The current work has been published in the March issue of Nature Medicine; for more information, please visit http://www.nature.com/nm/journal/v18/n3/full/nm.2699.html.

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