Category Archives: Stem Cells

Researchers at the Massachusetts General Hospital (MGH) have isolated egg producing stem cells from human ovarian tissues.

Scientists at the Vincent Center for Reproductive Biology, MGH used a special fluorescence-activated cell sorting protocol in mice models to grow new eggs from isolated stem cells.

The new study is seen as a precursor to solving fertility issues in women of reproductive ages. If made viable, the study could benefit young women undergoing cancer therapy and older women who have to resort to egg donors.

The current breakthrough challenges the widely-accepted notion that while men generate sperms throughout life, women are born with a fixed supply of eggs that deplete with age and are finally exhausted at menopause.

The current research, published in the March issue of Nature Medicine is a follow-up on an earlier landmark 2004 Nature paper suggesting that female mammals have the capability of producing egg cells throughout their adult reproductive stages.

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"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," said Jonathan Tilly, lead researcher and Director for Reproductive Biology at the MGH Vincent Department of Obstetrics and Gynecology.

To examine their arguments, the researchers injected green fluorescent protein (GFP)-labeled mouse oocyte-producing stem cells (OSCs) into the ovaries of normal adult mice. Several months later, the recipient mouse ovaries showed the presence of follicles containing oocytes (immature egg cells) with and without the marker protein. Similar GFP-labeled and unlabeled oocytes were observed in cell clusters flushed from the animals' oviducts after induced ovulation.

The GFP-labeled mouse eggs were fertilized in the lab to produce embryos that went on to the hatching blastocyst cell reproductive stages, indicating potential for normal egg cell development.

According to Tilly, "In this paper we provide the three key pieces of evidence requested by those who have been skeptical of our previous work."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", he added.

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Ovarian Stem Cells Holds Promise for Infertile Women: Study

February 28, 2012, 11:02 AM EST

By Ryan Flinn

Feb. 27 (Bloomberg) -- Stem cells taken from human ovaries were used to produce early-stage eggs by scientists in Boston who may have created a new method to help infertile women.

Females have a fixed number of eggs from birth that are depleted by the time of menopause. The finding, published today in the journal Nature Medicine, challenges the belief that their ovaries can’t make more. The research was led by Jonathan Tilly, the director of Massachusetts General Hospital’s Vincent Center for Reproductive Biology.

Tilly reported in 2004 that ovarian stem cells in mice create new eggs, or oocytes, in a way similar to how stem cells in male testes produce sperm throughout a man’s life. His latest work, if reproduced, would suggest the same is true for human ovaries, potentially pointing at new ways to aid fertility by delaying when the ovaries stop functioning.

“The 50-year-old belief in our field wasn’t actually based on data proving it was impossible, or not ongoing,” Tilly said in a telephone interview. “It was simply an assumption made because there was no evidence indicating otherwise. We have human cells that can produce new oocytes.”

In the study, healthy ovaries were obtained from consenting patients undergoing sex reassignment surgery. The researchers were able to identify ovarian stem cells because they express a rare protein that’s only seen in reproductive cells.

The stem cells from the ovaries were injected into human ovarian tissue that was then grafted under the skin of mice, which provided the blood supply that enabled growth. Within two weeks, early stage human follicles with oocytes had formed.

7-Million Eggs

A female is most endowed with oocytes, or eggs, as a fetus, when she has about 7 million. That number that drops to 1 million by birth, and around 300,000 by puberty. By menopause, the number is zero. Since the 1950’s, scientists thought that ovarian stem cells capable of producing new eggs are only active during fetal development.

“This paper essentially opens the door to the ability to control oocyte development in human ovaries,” Tilly said.

About 10 percent of women of child-bearing age in the U.S., or 6.1 million, have difficulty getting pregnant or staying pregnant, according to the Centers for Disease Control and Prevention. Most cases of female infertility are caused by problems with ovulation, hormone imbalance or age.

The study by Tilley and his colleagues offers “a new model system for understanding the human egg cell,” said David F. Albertini, director of the Center for Reproductive Services and professor in the department of molecular and integrative physiology at Kansas University, in a telephone interview.

‘Practical Applications”

Still, “there’s a long way to go before this has real practical applications. I’ve spent 35 years of my life studying egg cells and this is a cell that is at least as complicated as a neuron in the brain, if not more,” Albertini said.

The work needs to be reproduced and expanded by other scientists “to make it into something that will make us confident the cells are safe to use and we could actually use them to repopulate an egg-depleted ovary,” he said.

Tilly’s team is exploring the development of an ovarian stem-cell bank that can be cryogenically frozen and thawed without damage, unlike human eggs, he said. The researchers are also working to identify hormones and other growth factors for accelerating production of eggs from human ovarian stem cells and ways to improve in-vitro fertilization.

“The problem we face with IVF is we don’t have many eggs to work with,” he said. “These cells are renewable. If we are successful -- and it’s a big if -- in generating functioning eggs from these cells, we can generate as many eggs as we need to on a per patient basis.”

Tilly is also collaborating with researchers at the University of Edinburgh in the U.K. to determine whether the oocytes can be developed into fully mature human eggs for fertilizing. The U.S bans creating or fertilizing embryos for experimental purposes, he said.

A company Tilly co-founded, Boston-based OvaScience Inc., has licensed the technology for potential commercial applications.

--With assistance from Sarah Frier in New York. Editors: Angela Zimm, Andrew Pollack

To contact the reporter on this story: Ryan Flinn in San Francisco at rflinn@bloomberg.net

To contact the editor responsible for this story: Reg Gale at rgale5@bloomberg.net

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Ovarian Stem Cells Make Human Eggs in Possible Aid to Fertility

Left-hander C.J. Nitkowski appeared in 336 games, mostly in a relief role, over parts of 10 seasons. (AP)

The last known whereabouts of C.J. Nitkowski, in regard to his Major League career, was Washington, some seven years ago. It's entirely possible -- maybe even probable -- that this career is 100 percent over and out.

Yet there was Nitkowski on a field in the Dominican Republic last month, working in setup situations for Gigantes del Cibao in the middle of the Dominican Winter League's round-robin playoffs. Hoping, perhaps against hope, that some scout would take note of his new sidearm delivery and effectiveness against lefties and upper-80s gun readings and determine that he's worth another look. Hoping, basically, for one more chance -- a chance, he believes, that would have been completely out of the question if not for the blood-and-stem-cell treatments he received last summer. "From a medical standpoint," Nitkowski says, "[the treatments have] been a success. I'm healthy." But the route he took to get to this point is, in many ways, misunderstood. Nitkowski, who will turn 39 next week, received treatment identical to the one that picked Bartolo Colon's ailing arm and career off the scrap heap and made him a prominent member of the Yankees rotation last season. It's a treatment that, in Colon's case, caused a bit of an uproar in the headlines last summer, as such labels as "disputed" and "controversial" were used to describe it. In reality, though, the use of one's own stem cells to promote healing in an injured area is far from a new development. In fact, the microfracture procedure that is becoming more and more common in the treatment of knee injuries (it was performed on Victor Martinez last month) is, at its core, a stem-cell procedure. In microfracture, tiny holes are drilled in the bone to allow marrow to drip out and repair damaged tissue -- the mesenchymal stem cells inside the marrow provide the repairing power. In the cases of Nitkowski and Colon, the mesenchymal stem cells were extracted from bone marrow and from body fat and then injected into a blood-poor area -- Nitkowski's left shoulder and Colon's right shoulder and elbow -- to promote healing. Now, is this really a reliable way to treat an ailing athlete? That's a subject of scrutiny. Embryonic stem cells are the cause of controversy all their own, given the ethical and political debates over their use and concern by some members of the medical community that they have the potential to become cancerous tumors. With mesenchymal stem cells, on the other hand, the debate is not over morals or safety but, rather, efficacy. "There's very little evidence that bone marrow stem cells taken from one site and injected into another will do anything," Theodore Friedmann, a geneticist at the University of California at San Diego who heads the World Anti-Doping Agency's (WADA) gene doping panel, told ESPN The Magazine recently. "The most likely outcome is that if you put stem cells in places that are unfamiliar to them, like a knee or shoulder, most of them will just die." WADA initially banned all blood-spinning therapies before reversing its position in 2011 after studies failed to demonstrate that they enhance performance the way steroids do. So WADA currently has no position on the use of stem-cell treatments. In the face of skepticism, you have the case of Colon, who in the spring of 2010 was unsigned, unable to get any of his old velocity on his fastball and seemingly at the end of a once-dazzling career. Dr. James Purita, founder of the Institute of Regenerative and Molecular Orthopedics in Boca Raton, Fla., traveled to the Dominican Republic to perform platelet-rich plasma (PRP) and stem-cell treatments to help repair ligament damage in Colon's elbow and aid a torn rotator cuff. "There was a stigma that it was illegal, because we did it in the Dominican," Purita said. "But it was just because he lived there." And there was another stigma. Because Colon not only returned to the Majors but thrived in the first half of the 2011 season, some wondered if the procedure could be labeled a performance-enhancer. When Colon's story became public, Purita was questioned by Major League Baseball officials to ensure that he did not use human growth hormone in the procedure. Purita has admitted using HGH when treating non-athletes but said he knows better than to do so in these cases. MLB did a complete investigation, and no further action was taken. "We're not reinventing the wheel here," Purita said. "We've done a number of these procedures on people from all the major sports, with the exception of hockey. We've done some of the top players. But we keep it very discreet." The reason for the discretion, beyond the obvious HIPAA standards, is that some teams are leery of or reluctant to trust these stem-cell treatments. Purita said that he performed the procedure this winter on a Major League free agent who expressed an explicit desire that word not get out, because he didn't want it to affect his contract negotiations. However, the case of Colon, who signed a one-year, $2 million contract with the A's this winter, makes one wonder if biologic stem cells could be the next medical revolution, following arthroscopy and the ulnar collateral ligament replacement known as Tommy John surgery. Nitkowski, for one, hopes to find out. A member of eight Major League teams over parts of 10 seasons from 1995-2005, the left-handed Nitkowski appeared in 336 games, mostly in a relief role. In 2006 he began a five-year stint pitching for various teams in Asia -- first in the Japanese Pacific League, then in the Korean Baseball Organization. A year ago, hoping for another shot in the bigs, he began working on a sidearm delivery, only to injure his shoulder. It was around that time that Nitkowski heard about Colon. Intrigued, he made a call to Purita's office, and, within weeks he was in Boca Raton to undergo the procedure himself. Purita first drew fat from Nitkowski's waist, then drew bone marrow from the left side of Nitkowski's lower back. The liquids were spun in a centrifuge at 2,000 rotations per minute for about 15 minutes, isolating the platelets. They were then inserted into syringes and placed under an LED light for about 20 minutes -- a process that supposedly "kick starts" the cells inside. Once this process was complete, Purita injected the platelet-rich plasma and stem cells into Nitkowski's labrum and rotator cuff. This is an important distinction. Under U.S. Food and Drug Administration guidelines, mesenchymal stem cells must be "minimally manipulated," meaning they can't be harvested in a lab for days or weeks or transported elsewhere. "Everything," Purita said, "has to be used the same day, on the same patient, and everything has to be done at the point of care." In Nitkowski's case, the whole process took about four hours. Much to his amazement, he had full range of motion within 24 hours. "The rehab starts right away," he said. "You have to commit to it, like any injury. I never thought I had the mindset to do the tedious rehab work. But if you want it bad enough, you'll do it." Nitkowski's stem-cell treatment was performed in July of last year. And per the usual protocol in Purita's treatment plan, Nitkowski had a second PRP treatment four weeks later. By November he was throwing off a mound, and he was pleased with how his arm felt and how his sidearm-delivered stuff worked in the Dominican Republic last month. "I was sitting at 86, 87 [mph] and hitting 88-89," he said. "That's more than enough [velocity] from that arm angle." Though he hasn't pitched in the bigs in seven years, Nitkowski believes he could help a team, and he's hoping someone will give him a tryout in Spring Training.

Anthony Castrovince is a reporter for MLB.com. Read his columns and his blog, CastroTurf, and follow him on Twitter at @Castrovince. This story was not subject to the approval of Major League Baseball or its clubs.

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Nitkowski hoping stem cells lead to comeback

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.

Continued here:
Egg-making stem cells found in adult ovaries

(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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Stem cells in ovaries may grow new eggs, study shows

Contrary to the belief that women are born with a finite number of eggs, there may in fact be a way to replenish the supply, a new study suggests.

Researchers have isolated stem cells from adult human ovaries that appear to be capable of producing eggs.

The new findings follow a number of recent studies that have suggested such stem cells exist in adult mice, and can give rise to healthy offspring in animals that have had their fertility destroyed by chemotherapy. However, these studies have been controversial, because they go against years of research suggesting otherwise, experts say.

In the new study, the researchers devised a more rigorous way to isolate these cells, and for the first time, suggested their existence in people.

If true, the findings could have  implications for women's fertility treatments. Currently, women who choose to undergo in vitro fertilization (IVF) for infertility must endure hormone injections so doctors can retrieve eggs for fertilization, said study researcher Jonathan Tilly, director of the Vincent Center for Reproductive Biology at Massachusetts General Hospital. But if researchers could isolate egg-producing stem cells from ovaries, it might be possible to conduct that whole process outside the body, Tilly said.

"That whole program of IVF… becomes a non-necessity," Tilly said.

The study is published online today (Feb. 26) in the journal Nature Medicine.

Egg stem cells

In the new study, Tilly and colleagues isolated egg-producing stem cells from human ovary tissue by targeting a protein found on the surface of only these cells. In dishes, the cells grew into cells that had properties of human eggs. For instance, they had half the genetic material of other cells in the body.

Next, to show the stem cells could produce eggs, the researchers placed a gene into the stem cells that made them glow green, placed the stem cells into human ovarian tissue (taken during a biopsy), and grafted this tissue into mice. One to two weeks later, this tissue contained egg cells glowing green, showing they had formed from the stem cells, the researchers said.

The researchers don't yet know if these egg cells could be fertilized to produce children. The United States does not allow human eggs to be fertilized for research purposes. The researchers also don't know whether these egg-producing stem cells are active throughout a woman's life, or only when they receive a particular signal, Tilly said, although the researchers have a follow-up study planned to address this question.

The number of egg-producing stem cells appear to be quite minute. In mice, they make up about 0.014 percent of all cells in the ovary, Tilly said.

Still a controversy

"It's very novel and it's very exciting," said Dr. Sandra Carson, professor of obstetrics and gynecology, at Brown University's Women & Infants Hospital, who was not involved in the study.

"It certainly makes sense that there would be those stem cells still there," said Carson, noting men have stem cells that produce sperm throughout life.

However, other researchers say the new paper does not resolve the controversy of whether egg-producing cells exist in adult ovaries.

"I would like to see better characterization of this very small pool of cells that may be present in the ovary," said Dr. Marco Conti, professor and director of the Center for Reproductive Sciences at the University of California, San Francisco. Conti noted that some properties of the egg-producing cells described in this study do not match descriptions from previous studies.

And the paper still does not address whether these cells have any role in adult humans.

"There is no real functional evidence that this pool of cells indeed contributes to [egg formation] in the adult," Conti said.

But if these cells do in fact work in the way the researchers suspect, it might be possible to grow and mature them in an environment that resembles an ovary, Carson said.

In addition, unlike human eggs, these stem cells can be frozen without damage, Tilly said, so it may be possible to store them for future use.

Tilly is a co-founder of OvaScience, Inc, which has licensed the commercial potential of these findings for development of new fertility-enhancing procedures.

Pass it on:  Women's ovaries may contain stem cells that are capable of producing eggs after birth.

This story was provided by MyHealthNewsDaily, a sister site to LiveScience. Follow MyHealthNewsDaily staff writer Rachael Rettner on Twitter @RachaelRettner. Find us on Facebook.

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Stem Cells in Women's Ovaries May Produce New Eggs, Study Finds

Contrary to the belief that women are born with a finite number of eggs, there may in fact be a way to replenish the supply, a new study suggests.

Researchers have isolated stem cells from adult human ovaries that appear to be capable of producing eggs.

ANALYSIS: Old Genes Making Hulking Ants

The new findings follow a number of recent studies that have suggested such stem cells exist in adult mice, and can give rise to healthy offspring in animals that have had their fertility destroyed by chemotherapy. However, these studies have been controversial, because they go against years of research suggesting otherwise, experts say.

In the new study, the researchers devised a more rigorous way to isolate these cells, and for the first time, suggested their existence in people.

If true, the findings could have implications for women's fertility treatments. Currently, women who choose to undergo in vitro fertilization (IVF) for infertility must endure hormone injections so doctors can retrieve eggs for fertilization, said study researcher Jonathan Tilly, director of the Vincent Center for Reproductive Biology at Massachusetts General Hospital. But if researchers could isolate egg-producing stem cells from ovaries, it might be possible to conduct that whole process outside the body, Tilly said.

"That whole program of IVF… becomes a non-necessity," Tilly said.

The study is published online Feb. 26 in the journal Nature Medicine.

Egg Stem Cells

In the new study, Tilly and colleagues isolated egg-producing stem cells from human ovary tissue by targeting a protein found on the surface of only these cells. In dishes, the cells grew into cells that had properties of human eggs. For instance, they had half the genetic material of other cells in the body.

Next, to show the stem cells could produce eggs, the researchers placed a gene into the stem cells that made them glow green, placed the stem cells into human ovarian tissue (taken during a biopsy), and grafted this tissue into mice. One to two weeks later, this tissue contained egg cells glowing green, showing they had formed from the stem cells, the researchers said.

NEWS: Stems Cells Improve Vision in Two Blind Patients

The researchers don't yet know if these egg cells could be fertilized to produce children. The United States does not allow human eggs to be fertilized for research purposes. The researchers also don't know whether these egg-producing stem cells are active throughout a woman's life, or only when they receive a particular signal, Tilly said, although the researchers have a follow-up study planned to address this question.

The number of egg-producing stem cells appear to be quite minute. In mice, they make up about 0.014 percent of all cells in the ovary, Tilly said.

Still a Controversy

"It's very novel and it's very exciting," said Dr. Sandra Carson, professor of obstetrics and gynecology, at Brown University's Women & Infants Hospital, who was not involved in the study.

"It certainly makes sense that there would be those stem cells still there," said Carson, noting men have stem cells that produce sperm throughout life.

However, other researchers say the new paper does not resolve the controversy of whether egg-producing cells exist in adult ovaries.

"I would like to see better characterization of this very small pool of cells that may be present in the ovary," said Dr. Marco Conti, professor and director of the Center for Reproductive Sciences at the University of California, San Francisco. Conti noted that some properties of the egg-producing cells described in this study do not match descriptions from previous studies.

And the paper still does not address whether these cells have any role in adult humans.

"There is no real functional evidence that this pool of cells indeed contributes to [egg formation] in the adult," Conti said.

But if these cells do in fact work in the way the researchers suspect, it might be possible to grow and mature them in an environment that resembles an ovary, Carson said.

In addition, unlike human eggs, these stem cells can be frozen without damage, Tilly said, so it may be possible to store them for future use.

Tilly is a co-founder of OvaScience, Inc, which has licensed the commercial potential of these findings for development of new fertility-enhancing procedures.

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Stem Cells in Ovaries May Give Women More Eggs

POSTED: 1:07 pm EST February 26, 2012

WASHINGTON -- For 60 years, doctors have believed women were born with all the eggs they'll ever have. Now Harvard scientists are challenging that dogma, saying they've discovered the ovaries of young women harbor very rare stem cells capable of producing new eggs.If Sunday's report is confirmed, harnessing those stem cells might one day 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," said lead researcher Jonathan Tilly of Harvard's Massachusetts General Hospital, who has long hunted these cells in a series of controversial studies.Tilly's previous work drew fierce skepticism, and independent experts urged caution about the latest findings.A key next step is to see whether other laboratories can verify the work. If so, then it would take years of additional research to learn how to use the cells, said Teresa Woodruff, fertility preservation chief at Northwestern University's Feinberg School of Medicine.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," said David Albertini, director of the University of Kansas' Center for Reproductive Sciences. 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.But do they exist in women? Enter the new work, reported Sunday in the journal Nature Medicine.First Tilly had to find healthy human ovaries to study. He collaborated with scientists at Japan's Saitama Medical University, who were freezing ovaries donated for research by healthy 20-somethings who underwent a sex-change operation.Tilly also had to address a criticism: How to tell if he was finding true stem cells or just very immature eggs. His team latched onto a protein believed to sit on the surface of only those purported stem cells and fished them out. To track what happened next, the researchers inserted a gene that makes some jellyfish glow green into those cells. If the cells made eggs, those would glow, too."Bang, it worked - cells popped right out" of the human tissue, Tilly said.Researchers watched through a microscope as new eggs grew in a lab dish. Then came the pivotal experiment: They injected the stem cells into pieces of human ovary. They transplanted the human tissue under the skin of mice, to provide it a nourishing blood supply. Within two weeks, they reported telltale green-tinged egg cells forming.That's still a long way from showing they'll mature into usable, quality eggs, Albertini said.And 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.Further down the road, he wonders if it also might be possible to recharge an aging woman's ovaries.The new research was funded largely by the National Institutes of Health. Tilly co-founded a company, OvaScience Inc., to try to develop the findings into fertility treatments.

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Report: Women Have Rare Egg-Producing Stem Cells

Study Suggests Women May Be Able to Make More Eggs as They Age

Feb. 26, 2012 -- Scientists say they have found a way to use ovarian stem cells to perhaps one day help infertile women get pregnant -- or add years to a woman’s reproductive cycle.

In a study published in Nature Medicine, researchers report finding egg-producing stem cells in human ovaries. They also report being able to make some of those ovarian stem cells grow into immature eggs that may someday be useful for reproduction.

At this point, such “seed” eggs can’t be fertilized by sperm. But if scientists are able to entice them to mature and can prove they can be fertilized and grow into embryos -- a feat that has been reported in mice -- it would overturn a long-held scientific belief that women can’t make new eggs as they get older.

“What it does is really open a door into human reproduction that 10 years ago didn’t even exist,” says researcher Jonathan L. Tilly, PhD, director of the Vincent Center for Reproductive Biology at Massachusetts General Hospital, in Boston.

Outside experts agree. They say the findings could have profound importance for reproductive medicine and aging, allowing doctors not only to restore a woman’s fertility but also to potentially delay menopause.

“I think the significance of this work is like reporting that we found microorganisms on Mars,” says Kutluk Oktay, MD, who directs the Division of Reproductive Medicine and the Institute for Fertility Preservation at New York Medical College in Valhalla, N.Y.

Still, It’s a Long Way to Mars

“It’s a proof of principle that they could do it,” says David F. Albertini, PhD, director of the Center for Reproductive Sciences at the University of Kansas Medical Center in Kansas City, Kan.

“The world wants to know today if we’re ready to restore fertility in women, whether they’ve aged or been treated for cancer or whatever,” Albertini says, adding that he doesn’t think that’s on the horizon. “This is an extremely rare event, at best.”

The egg-generating stem cells the researchers were able to extract from ovaries were very rare. The researchers only came across one for every 10,000 or so ovarian cells that they counted.

But when they took those cells and implanted them back into human ovarian tissue, they divided and essentially made young eggs.

Tilly says his team stopped short of trying to make one of the eggs functional because “for a lot of reasons, as it should be,” it is illegal in the U.S. to experimentally fertilize human eggs.

“We think the evidence provided clearly indicates that this very unique, newly discovered pool of cells does exist in women,” he says.

A Lot of Potential

“It’s a really exciting result,” says Evelyn Telfer, PhD, a cell biology expert at the University of Edinburgh in Scotland.

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Future Fertility Fix? Egg-Producing Stem Cells Found in Human Ovaries

Public release date: 26-Feb-2012
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Contact: Sue McGreevey
smcgreevey@partners.org
617-724-2764
Massachusetts General Hospital

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. Tilly is a co-founder of OvaScience, Inc. (www.ovascience.com), which has licensed the commercial potential of these and other patent-protected findings of the MGH-Vincent team for development of new fertility-enhancing procedures.

Massachusetts General Hospital (www.massgeneral.org), founded in 1811, is the original and largest teaching hospital of Harvard Medical School. The MGH conducts the largest hospital-based research program in the United States, with an annual research budget of more than $750 million and major research centers in AIDS, cardiovascular research, cancer, computational and integrative biology, cutaneous biology, human genetics, medical imaging, neurodegenerative disorders, regenerative medicine, reproductive biology, systems biology, transplantation biology and photomedicine.

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Mass. General researchers isolate egg-producing stem cells from adult human ovaries