ScienceDaily (Feb. 26, 2012) — For the first time, Massachusetts General Hospital (MGH) researchers have isolated egg-producing stem cells from the ovaries of reproductive age women and shown these cells can produce what appear to be normal egg cells or oocytes. In the March issue of Nature Medicine, the team from the Vincent Center for Reproductive Biology at MGH reports the latest follow-up study to their now-landmark 2004 Nature paper that first suggested female mammals continue producing egg cells into adulthood.

"The primary objective of the current study was to prove that oocyte-producing stem cells do in fact exist in the ovaries of women during reproductive life, which we feel this study demonstrates very clearly," says Jonathan Tilly, PhD, director of the Vincent Center for Reproductive Biology in the MGH Vincent Department of Obstetrics and Gynecology, who led the study. "The discovery of oocyte precursor cells in adult human ovaries, coupled with the fact that these cells share the same characteristic features of their mouse counterparts that produce fully functional eggs, opens the door for development of unprecedented technologies to overcome infertility in women and perhaps even delay the timing of ovarian failure."

The 2004 report from Tilly's team challenged the fundamental belief, held since the 1950s, that female mammals are born with a finite supply of eggs that is depleted throughout life and exhausted at menopause. That paper and a 2005 follow-up published in Cell showing that bone marrow or blood cell transplants could restore oocyte production in adult female mice after fertility-destroying chemotherapy were controversial; but in the intervening years, several studies from the MGH-Vincent group and other researchers around the world have supported Tilly's work and conclusions.

These supporting studies include a 2007 Journal of Clinical Oncology report from the MGH-Vincent team that showed female mice receiving bone marrow transplants after oocyte-destroying chemotherapy were able to have successful pregnancies, delivering pups that were their genetic offspring and not of the marrow donors. A 2009 study from a team at Shanghai Jiao Tong University in China, published in Nature Cell Biology, not only isolated and cultured oocyte-producing stem cells (OSCs) from adult mice but also showed that those OSCs, after transplantation into the ovaries of chemotherapy-treated female mice, gave rise to mature oocytes that were ovulated, fertilized and developed into healthy offspring.

"That study singlehandedly deflated many of the arguments from critics of our earlier Nature paper by showing that oocyte-producing stem cells exist in mice and could develop into fully functional eggs," says Tilly. Another paper from a west-coast biotechnology company, published in Differentiation in 2010, provided further independent confirmation of Tilly's earlier conclusions regarding the presence of oocyte-producing stem cells in ovaries of adult mice.

Tilly is quick to point out, however, "These follow-up studies, while providing definitive evidence that oocyte-producing stem cells exist in ovaries of adult female mammals, were not without their limitations, leaving the question open in some scientific circles of whether the adult oocyte pool can be renewed. For example, the protocol used to isolate OSCs in the 2009 Nature Cell Biology study is a relatively crude approach that often results in the contamination of desired cells by other cell types." To address this, the MGH-Vincent team developed and validated a much more precise cell-sorting technique to isolate OSCs without contamination from other cells.

The 2009 study from China also had isolated OSCs based on cell-surface expression of a marker protein called Ddx4 or Mvh, 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. Using their state-of-the-art fluorescence-activated cell sorting techniques, the MGH-Vincent team 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 investigators 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, which Tilly considers to be the most groundbreaking, 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, a professor of Obstetrics, Gynecology and Reproductive Biology at Harvard Medical School and chief of Research at the MGH Vincent Department of Obstetrics and Gynecology. "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," he adds. "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."

Among the many potential clinical applications for these findings that Tilly's team is currently exploring are 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, and other approaches to improve the outcomes of IVF and other infertility treatments.

Tilly notes that an essential part of his group's accomplishment was collaboration with study co-author Yasushi Takai, MD, PhD, a former MGH research fellow on Tilly's team and now a faculty member at Saitama Medical University in Japan. Working with his clinical colleagues at Saitama, Takai was able to provide healthy ovarian tissue from consenting patients undergoing sex reassignment surgery, many in their 20s and early 30s. Co-lead authors of the Nature Medicine report are Yvonne White, PhD, and Dori Woods, PhD, of the Vincent Center for Reproductive Biology at MGH. Additional co-authors are Osamu Ishihara, MD, PhD, and Hiroyuki Seki, MD, PhD, of Saitama Medical University.

The study was supported by a 10-year MERIT Award to Tilly from the National Institute on Aging, a Ruth L. Kirschstein National Research Service Award from the National Institutes of Health, the Henry and Vivian Rosenberg Philanthropic Fund, the Sea Breeze Foundation, and Vincent Memorial Hospital Research Funds.

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The above story is reprinted from materials provided by Massachusetts General Hospital.

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Journal Reference:

Yvonne A R White, Dori C Woods, Yasushi Takai, Osamu Ishihara, Hiroyuki Seki, Jonathan L Tilly. Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women. Nature Medicine, 2012; DOI: 10.1038/nm.2669

Note: If no author is given, the source is cited instead.

Disclaimer: This article is not intended to provide medical advice, diagnosis or treatment. Views expressed here do not necessarily reflect those of ScienceDaily or its staff.

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Egg-producing stem cells isolated from adult human ovaries

23-02-2012 19:48 Complete video at fora.tv Dr. Bruce Conklin, Senior Investigator at the Gladstone Institute of Cardiovascular Disease, discusses the potential applications of stem cells in personalized medicine. By introducing a set of genes called the "Yamanaka factors," a simple skin sample can be transformed into something as complex as functioning heart tissue -- which could potentially be used to completely personalize medical testing. ---- Stem cells have the remarkable potential to develop into many different cell types in the body during early life and growth. In many tissues, they serve as a sort of internal repair system, dividing essentially without limit to replenish other cells as long as the person or animal is still alive. San Francisco's Gladstone Institutes is a leading force in stem cell research. In this presentation, Gladstone Investigator Dr. Bruce Conklin explains the surprising past, present, and future of stem cells.

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Stem Cells and Medicine: Be Your Own Guinea Pig - Video

24-02-2012 16:02 Stem cells from the ovaries of reproductive age women can give rise to cells that appear to be mature oocytes, suggesting that women can produce more eggs than the batch they are born with. The findings, reported in the March 2012 issue of Nature Medicine, open the door to a new generation of assisted fertility treatments. http://www.nature.com

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Stem cells put women on fertile ground - by Nature Video - Video

In a new study, Harvard researchers say they have found stem cells in women that can be used to grow new eggs. Not surprisingly, this has raised much discussion about whether a woman’s biological clock can be stopped – why worry about running out of eggs if you can just make new ones whenever you  need them?

The work described in the paper, published online Sunday by the journal Nature Medicine, is still a long way from being useful to women in need of fertility treatments. And many scientists remain skeptical that these ovarian stem cells really can mature into healthy eggs.

But as long as we’re in the pie-in-the-sky realm, let’s consider another way that the ability to grow an abundant supply of eggs would be helpful: to make human embryonic stem cell lines that would be perfectly matched to patients.

This was a hot area of research in the middle of the last decade. While many scientists studied stem cells made from embryos that were no longer needed for fertility treatments, a smaller group was pursuing a derivation method called somatic cell nuclear transfer, or SCNT. It’s better known as “therapeutic cloning.”

Here’s the idea: You take an egg and remove all the DNA in the cell nucleus. Then you replace it with DNA from a patient. You give the egg an electric shock so it starts dividing and growing into an early-stage embryo.

But instead of implanting this embryo into a uterus and producing a baby that’s a genetic copy of another person, you would use it to make a line of human embryonic stem cells. Then you can use those stem cells to make replacement parts – new cardiac cells for patients who suffered heart attacks, for instance, or nerve cells that would replace those lost after a spinal cord injury. In theory, the new cells would work perfectly because they’d be a perfect genetic match. This is the vision of regenerative medicine.

For a few months back in 2005, it looked like this vision was on the verge of reality. South Korean researcher Hwang Woo Suk published a landmark paper in the journal Science in which he claimed to have made 11 lines of stem cells that were genetic matches to patients with Type 1 diabetes, spinal cord injuries and the so-called Bubble Boy disease. Scientists rejoiced, as did doctors and patients. But a few months later, Hwang was accused of faking the results. The study was retracted, and Hwang was prosecuted for embezzling research money and violating ethics laws.

Since then, researchers at Oregon Health & Sciences University have managed to clone monkey embryos in order to create embryonic stem cells. But in a 2007 study in the journal Nature, they said they had to use 304 eggs to make just two viable cell lines.

It’s hard to imagine how scientists would ever get their hands on 304 human eggs, especially since they generally aren’t allowed to pay women who might be willing to donate eggs for research purposes. It’s also not clear that a few hundred eggs would be enough to guarantee at least one line of stem cells. South Korean investigators eventually discovered that Hwang used 2,236 eggs in his studies that failed to produce a single embryo.

This is one of the major reasons why SCNT studies fell by the wayside. (For more on that, read this story from 2006.) But if there were a relatively simple way to grow hundreds – or thousands – of eggs in the lab, some scientists are confident they could create stem cells through therapeutic cloning.

If so, that would make the research at Harvard relevant to a whole lot of people besides women who hear their biological clocks ticking.

A summary of the Nature Medicine study is online here.

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Eggs made from stem cells could treat more than just fertility

(CBS/AP) Stem cells in young women's ovaries are capable of producing new eggs, according to a new study. The findings challenge 60 years of dogma that women are born with all the eggs they'll ever have.

PICTURES: Human eggs: 9 fascinating facts

For the study, published in the Feb. 26 issue of Nature Medicine and led by Jonathan Tilly of Massachusetts General Hospital, researchers examined healthy human ovaries donated by 20-something Japanese women who were undergoing a sex-change operation. The researchers fished out stem cells by searching for a protein found only on the surface of stem cells. The researchers then injected those stem cells into pieces of human ovary, transplanting the tissue under the skin of mice, to provide the tissue with a nourishing blood supply.

What happened? New egg cells formed within two weeks.

That's still a long way from showing they'll mature into usable, quality eggs, David Albertini, director of the University of Kansas' Center for Reproductive Sciences, cautioned.

Still, these findings could lead to better treatments for women left infertile because of disease - or simply because they're getting older.

"Our current views of ovarian aging are incomplete. There's much more to the story than simply the trickling away of a fixed pool of eggs," Tilly, who has long hunted these cells in a series of controversial studies, said.

Tilly's previous work has drawn skepticism, and independent experts urged caution about the latest findings, so the next step is to see whether other laboratories can verify the work. If the findings are confirmed, then it would take years of additional research to learn how to use the cells, Teresa Woodruff, fertility preservation chief at Northwestern University's Feinberg School of Medicine, said.

"This is experimental," Dr. Avner Hershlag, chief of the Center for Human Reproduction at North Shore-LIJ Health System in Manhasset, N.Y., told HealthDay. He said the study is "exciting" but emphasized the work is still very preliminary. "This is a beginning of perhaps something that could bring in new opportunities, but it's going to be a long time in my estimation until clinically we'll be able to actually have human eggs created from stem cells that make babies."

Still, even a leading critic said such research may help dispel some of the enduring mystery surrounding how human eggs are born and mature.

"This is going to spark renewed interest, and more than anything else it's giving us some new directions to work in," Albertini said. While he has plenty of questions about the latest work, "I'm less skeptical," he said.

Scientists have long taught that all female mammals are born with a finite supply of egg cells, called ooctyes, that runs out in middle age. Tilly, Mass General's reproductive biology director, first challenged that notion in 2004, reporting that the ovaries of adult mice harbor some egg-producing stem cells. Recently, Tilly noted, a lab in China and another in the U.S. also have reported finding those rare cells in mice.

More work is needed to tell exactly what these cells are, cautioned reproductive biologist Kyle Orwig of the University of Pittsburgh Medical Center, who has watched Tilly's work with great interest.

But if they're really competent stem cells, Orwig asked, then why would women undergo menopause? Indeed, something so rare wouldn't contribute much to a woman's natural reproductive capacity, added Northwestern's Woodruff.

Tilly argues that using stem cells to grow eggs in lab dishes might one day help preserve cancer patients' fertility. Today, Woodruff's lab and others freeze pieces of girls' ovaries before they undergo fertility-destroying chemotherapy or radiation. They're studying how to coax the immature eggs inside to mature so they could be used for in vitro fertilization years later when the girls are grown. If that eventually works, Tilly says stem cells might offer a better egg supply.

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Study: Stem cells in ovaries may grow new eggs

Scientists might have found a way for a woman to be able to produce more eggs, potentially aiding and extending her fertility. The study, published in the journal Nature Medicine, found the ovaries of young women might still contain egg-producing stem cells, according to a report by MSNBC.

How could these stem cells potentially be used?

Theoretically, they could be used to develop new treatments for women who are struggling with infertility issues. The lead researcher on the study, Jonathan Tilly, has said that the stem cells could potentially be used to preserve the fertility of younger women who have struggled with serious diseases, like cancer, that may require harsh treatments that destroy the viability of their available eggs. He also speculated that they may be able to be used to restore egg production for an older woman that is no longer fertile.

What did the study involve?

Tilly, who works through Harvard-affiliated Massachusetts General Hospital, had first discovered these stem cells in mice. He then went looking for them in donated ovaries that he acquired through a partnership with a Japanese hospital.

The stem cells had to be isolated in order for Tilly to test them for their ability to produce new eggs. After being injected with a gene that would change them to a particular color, the stem cells were placed in part of a human ovary and grafted under the skin of mice to monitor the effect, according to My Health News Daily. The grafted stem cells did in fact appear to begin to grow new, albeit immature, eggs.

What are the potential challenges facing this study?

Mostly, skepticism. Some experts that have reviewed the study, including Dr. Mario Conti of the Center for Reproductive Sciences at the University of California, San Francisco, have pointed out that Tilly has failed to prove that these cells can be used to grow eggs in humans rather than mice. Other criticism concerns the stem cells themselves, which appear to make up a very small amount of the cells of the ovaries, and whether or not they are capable of producing a mature egg that can be fertilized and grow into a human being.

What are the next research steps?

Tilly plans on conducting more studies to test the potential of these stem cells. WebMD reported that he has already partnered with cell biologist Dr. Evelyn Telfer at the University of Edinburgh in Scotland to begin developing techniques to take the immature, or "seed" eggs and encourage them to become fully-mature eggs which may be able to be used.

Tilly and others have cautioned that his research is just the first step in a long journey. Any practical application for his research is still years away.

Vanessa Evans is a musician and freelance writer based in Michigan, with a lifelong interest in health and nutrition issues.

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Stem Cells Might Have the Potential to Produce New Eggs

It’s time to rewrite the textbooks. For 60 years, everyone from high-school biology teachers to top fertility specialists has been operating under the assumption that women are born with all the eggs they will ever produce, with no way to replenish that supply. But the discovery of human egg-producing stem cells, harvested from the ovaries of six women aged 22 to 33, puts that dogma in doubt.

The work, published online in Nature Medicine1 by Jonathan Tilly and colleagues at Massachusetts General Hospital in Boston, parallels the findings of a Shanghai-based group2 that isolated similar stem cells from mice in 2009. However, both this and Tilly’s earlier work in mice3 remained controversial, with many experts sceptical that such stem cells existed.

“This is unequivocal proof that not only was the mouse biology correct, but what we proposed eight years ago was also correct — that there was a human population of stem cells in young adult tissue,” says Tilly.

To address the doubts, Tilly’s team began by developing a more sensitive method for identifying and collecting mouse ovarian stem cells. Their method, based on a technique called fluorescence-activated cell sorting (FACS), attaches a fluorescently labelled antibody to a protein, Ddx4, that is present on the outer surface of the stem cells but not on the surface of the later-stage egg cells or oocytes. The FACS instrument lines up cells in single file and sorts them one by one, separating the labelled ones from the rest; it also gets rid of dead or damaged cells, such as oocytes, in which internal Ddx4 might become accessible to the antibody. This method is more selective than previous isolation methods, which did not get rid of such cells.

Once the team confirmed that it had isolated mouse ovarian stem cells by this method, it set its sights on reproductive-age human ovaries. Yasushi Takai, a former research fellow in Tilly’s lab and now a reproductive biologist at Saitama Medical University in Japan, supplied frozen whole ovaries removed from sex-reassignment patients, all young women of reproductive age. “It was 9 November when we did the first human FACS sort and I knew immediately that it had worked,” says Tilly. “I cannot even put into words the excitement — and, to some degree, the relief — I felt.”

The cells they pulled out, called oogonial stem cells (OSCs), spontaneously generated apparently normal immature oocytes when cultured in the lab. To look at the development of the putative human OSCs in a more natural environment, the team labelled the cells with green fluorescent protein to make them traceable, and injected them into fragments ofadulthumanovarian tissue, which were then transplanted under the skin of mice. After one to two weeks of growth, the OSCs had formed green-glowing cells that looked like oocytes and that also expressed two of the genetic hallmarks of this cell type.

“There’s no confirmation that we have baby-making eggs yet, but every other indication is that these cells are the real deal — bona fide oocyte precursor cells,” says Tilly. The next step, to test whether the human OSC-derived oocytes can be fertilized and form an early embryo, will require special considerations — namely, private funding to support the work in the United States (federal funding cannot by law be used for any research that will result in the destruction of a human embryo, whatever the source of the embryo) or a licence from the UK Human Fertilisation and Embryology Authority to do the work with collaborators in the United Kingdom.

“I’ve seen these cells and how they behave. They’re convincing and impressive.”

Evelyn Telfer, a reproductive biologist at the University of Edinburgh, UK, was once sceptical of the mouse work, but has become a believer. “I’ve visited [Tilly’s] lab, seen these cells and how they behave. They’re convincing and impressive,” she says. Telfer, who studies the maturation of human eggs in vitro, will work with Tilly to try to grow the OSC-derived eggs to the point at which they are ready for fertilization.

She notes that there’s still no evidence that the OSCs form new eggs naturally in the body. However, if they could be coaxed in a dish to make eggs that could successfully be used for in vitro fertilization (IVF), it would change the face of assisted reproduction.

“That’s a huge ‘if’,” admits Tilly. But, he continues, it could mean an unlimited supply of eggs for women who have ovarian tissue that still hosts OSCs. This group could include cancer patients who have undergone sterilizing chemotherapy, women who have gone through premature menopause, or even those experiencing normal ageing. Tilly says that follow-up studies have confirmed that OSCs exist in the ovaries of women well into their 40s.

In addition, growing eggs from OSCs in the lab would allow scientists to screen for hormones or drugs that might reinvigorate these cells to keep producing eggs in the body and slow down women’s biological clocks. “Even if you could gain an additional five years of ovarian function, that would cover most women affected by IVF,” notes Tilly.

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Egg-making stem cells found in adult ovaries