Lab-Made Egg and Sperm Precursors Raise Prospect for Infertility Treatment.


A technical tour de force, which involved creating primordial germ cells from mouse skin cells, is prompting scientists to consider attempting this experiment with human cells

Since last October, molecular biologist Katsuhiko Hayashi has received around a dozen e-mails from couples, most of them middle-aged, who are desperate for one thing: a baby. One menopausal woman from England offered to come to his laboratory at Kyoto University in Japan in the hope that he could help her to conceive a child. “That is my only wish,” she wrote.

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The requests started trickling in after Hayashi published the results of an experiment that he had assumed would be of interest mostly to developmental biologists. Starting with the skin cells of mice in vitro, he created primordial germ cells (PGCs), which can develop into both sperm and eggs. To prove that these laboratory-grown versions were truly similar to naturally occurring PGCs, he used them to create eggs, then used those eggs to create live mice. He calls the live births a mere ‘side effect’ of the research, but that bench experiment became much more, because it raised the prospect of creating fertilizable eggs from the skin cells of infertile women. And it also suggested that men’s skin cells could be used to create eggs, and that sperm could be generated from women’s cells. (Indeed, after the research was published, the editor of a gay and lesbian magazine e-mailed Hayashi for more information.)

Despite the innovative nature of the research, the public attention surprised Hayashi and his senior professor, Mitinori Saitou. They have spent more than a decade piecing together the subtle details of mammalian gamete production and then recreating that process in vitro — all for the sake of science, not medicine. Their method now allows researchers to create unlimited PGCs, which were previously difficult to obtain, and this regular supply of treasured cells has helped to drive the study of mammalian reproduction. But as they push forward with the scientifically challenging transition from mice to monkeys and humans, they are setting the course for the future of infertility treatments — and perhaps even bolder experiments in reproduction. Scientists and the public are just starting to grapple with the associated ethical issues.

“It goes without saying that [they] really transformed the field in the mouse,” says Amander Clark, a fertility expert at the University of California, Los Angeles. “Now, to avoid derailing the technology before it’s had a chance to demonstrate its usefulness, we have to have conversations about the ethics of making gametes this way.”

Back to the beginning
In the mouse, germ cells emerge just after the first week of embryonic development, as a group of around 40 PGCs. This little cluster goes on to form the tens of thousands of eggs that female mice have at birth, and the millions of sperm cells that males produce every day, and it will pass on the mouse’s entire genetic heritage. Saitou wanted to understand what signals direct these cells throughout their development.

Over the past decade, he has laboriously identified several genes — including Stella,Blimp1 and Prdm14 — that, when expressed in certain combinations and at certain times, play a crucial part in PGC development. Using these genes as markers, he was able to select PGCs from among other cells and study what happens to them. In 2009, from experiments at the RIKEN Center for Developmental Biology in Kobe, Japan, he found that when culture conditions are right, adding a single ingredient — bone morphogenetic protein 4 (Bmp4) — with precise timing is enough to convert embryonic cells to PGCs. To test this principle, he added high concentrations of Bmp4 to embryonic cells. Almost all of them turned into PGCs. He and other scientists had expected the process to be more complicated.

Saitou’s approach — meticulously following the natural process — was in stark contrast to work that others were doing, says Jacob Hanna, a stem-cell expert at the Weizmann Institute of Science in Rehovot, Israel. Many scientists try to create specific cell types in vitro by bombarding stem cells with signalling molecules and then picking through the resulting mixture of mature cells for the ones they want. But it is never clear by what process these cells are formed or how similar they are to the natural versions. Saitou’s efforts to find out precisely what is needed to make germ cells, to get rid of superfluous signals and to note the exact timing of various molecules at work, impressed his colleagues. “There’s a really beautiful hidden message in this work — that differentiation of cells [in vitro] is really not easy,” says Hanna. Harry Moore, a stem-cell biologist at the University of Sheffield, UK, regards the careful recapitulation of germ-cell development as “a triumph”.

Until 2009, Saitou’s starting point had been cells taken from a live mouse epiblast — a cup-like collection of cells lining one end of the embryo that forms at the end of the first week of development, just before the PGCs emerge. But to truly master the process, Saitou wanted to start with readily available, cultured cells.

That was a project for Hayashi, who in 2009 had returned to Japan from the University of Cambridge, UK, where, like Saitou before him, he had completed a four-year stint in the laboratory of a pioneer in the field, Azim Surani. Surani speaks highly of the two scientists, saying that they “complement each other in temperament and in their style and approach to solving problems”. Saitou is “systematic” and “single-minded about setting and accomplishing his objectives”, whereas Hayashi “works more intuitively, and takes a broader view of the subject and has outwardly a more relaxed approach”, he says. “Together they form a very strong team indeed.”

Hayashi joined Saitou at Kyoto University, which he quickly found was different from Cambridge. There was much less time spent on theoretical discussions than Hayashi was used to; instead, one jumped into experiments. “In Japan we just do it. Sometimes that can be very inefficient, but sometimes it makes a huge success,” he says.

Hayashi tried to use epiblast cells — Saitou’s starting point — but instead of using extracted cells as Saitou did, he tried to culture them as a stable cell line that could produce PGCs. That did not work. Hayashi then drew on other research showing that one key regulatory molecule (activin A) and a growth factor (basic fibroblast growth factor) could convert cultured early embryonic stem cells into cells akin to epiblasts. That sparked the idea of using these two factors to induce embryonic stem cells to differentiate into epiblasts, and then to apply Saitou’s previous formula to push these cells to become PGCs. The approach was successful.

To prove that these artificial PGCs were faithful copies, however, they had to be shown to develop into viable sperm and eggs. The process by which this happens is complicated and ill understood, so the team left the job to nature — Hayashi inserted the PGCs into the testes of mice that were incapable of producing their own sperm, and waited to see whether the cells would develop. Saitou thought that it would work, but fretted. “It seemed like a 50/50 chance,” he says. “We were excited and worried at the same time.” But, on the third or fourth mouse, they found testes with thick, dark seminiferous tubules, stuffed with sperm. “It happened so properly. I knew they would generate pups,” says Hayashi. The team injected these sperm into eggs and inserted the embryos into female mice. The result was fertile males and females.

They repeated the experiment with induced pluripotent stem (iPS) cells — mature cells that have been reprogramed to an embryo-like state. Again, the sperm were used to produce pups, proving that they were functional — a rare accomplishment in the field of stem-cell differentiation, where scientists often argue over whether the cells that they create are truly what they seem to be. “This is one of the few examples in the entire field of pluripotent-stem-cell research where a fully functional cell type has been unequivocally generated starting from a pluripotent stem cell in a dish,” says Clark.

They expected eggs to be more complex, but last year, Hayashi made PGCs in vitrowith cells from a mouse with normal coloring and then transferred them into the ovaries of an albino mouse. The resulting eggs were fertilized in vitro and implanted into a surrogate. “I knew it had worked,” he says, when he saw the pups’ dark eyes pressing through their translucent eyelids.

Germ-cell bounty
Other researchers have been able to replicate the process to generate laboratory-grown PGCs (although none contacted by Nature had used them to produce liveanimals). Artificial PGCs are of particular use to scientists who study epigenetics: the biochemical modifications to DNA that determine which genes are expressed. These modifications — most often the addition of methyl groups to individual DNA bases — in some instances carry a sort of historical record of what an organism has experienced (for example, exposure to foreign chemicals in the womb). In a similar way to how they work in other cells, epigenetic markers push PGCs to their fate during embryonic development, but PGCs are unique because when they develop into sperm and eggs, the epigenetic markers are erased. This allows the cells to create a new zygote that is capable of forming all cell types.

Faults in subtle epigenetic changes are expected to contribute to infertility and the emergence of disorders such as testicular cancer. Already, Surani’s and Hanna’s groups have used the artificial PGCs to investigate the role of individual enzymes in epigenetic regulation, which may one day show how the epigenetic networks are involved in disease.

Indeed, the in vitro-generated PGCs offer millions of cells for scientists to study, instead of the 40 or so that can be obtained by dissecting early embryos, says Hanna. “This is a big deal because here we have these rare cells — PGCs — that are undergoing dramatic genome-wide epigenetic changes that we barely understand,” he says. “The in vitro model has provided unprecedented accessibility to scientists,” agrees Clark.

Clinical relevance
But Hayashi and Saitou have little to offer to the infertile couples begging for their help. Before this protocol can be used in the clinic, there are large wrinkles to be ironed out.

Saitou and Hayashi have found that although the offspring generated by their technique usually seem to be healthy and fertile, the PGCs that these offspring generate in turn are not completely ‘normal’. The second-generation PGCs often produce eggs that are fragile, misshapen and sometimes dislodged from the complex of cells that supports them. When fertilized, the eggs often divide into cells with three sets of chromosomes rather than the normal two, and the rate at which the artificial PGCs successfully produce offspring is only one-third of the rate for normal in vitrofertilization (IVF). Yi Zhang, who studies epigenetics at Harvard Medical School in Boston, Massachusetts, and who has been using Saitou’s method, has also found thatin vitro PGCs do not erase their previous epigenetic programming as well as naturally occurring PGCs. “We have to be aware that these are PGC-like cells and not PGCs,” he says.

In addition, two major technical challenges remain. The first is working out how to make the PGCs convert to mature sperm and eggs without transplanting them back into testes or ovaries; Hayashi is trying to decipher the signals that ovaries and testes give to the PGCs that tell them to become eggs or sperm, which he could then add to artificial PGCs in culture to lead them through these stages.

But the most formidable challenge will be repeating the mouse PGC work in humans. The group has already started tweaking human iPS cells using the same genes that Saitou pinpointed as being important in mouse germ-cell development, but both Saitou and Hayashi know that human signalling networks are different from those in mice. Moreover, whereas Saitou had ‘countless’ numbers of live mouse embryos to dissect, the team has no access to human embryos. Instead, the researchers receive 20 monkey embryos per week from a nearby primate facility, under a grant of ¥1.2 billion (US$12 million) over five years. If all goes well, Hayashi says, they could repeat the mouse work in monkeys within 5–10 years; with small tweaks, this method could then be used to produce human PGCs shortly after.

But making PGCs for infertility treatment will still be a huge jump, and many scientists — Saitou included — are urging caution. Both iPS and embryonic stem cellsfrequently pick up chromosomal abnormalities, genetic mutations and epigenetic irregularities during culture. “There could be potentially far-reaching, multi-generational consequences if something went wrong in a subtle way,” says Moore.

Proof that the technique is safe in monkeys would help to allay concerns. But how many healthy monkeys would need to be born before the method could be regarded as safe? And how many generations should be observed?

Eventually, human embryos will need to be made and tested, a process that will be slowed by restrictions on creating embryos for research. New, non-invasive imaging techniques will enable doctors to sort good from bad embryos with a high degree of accuracy. Embryos that seem to be similar to normal IVF embryos could get the go-ahead for implantation into humans. This might happen with private funding or in countries with less-restrictive attitudes towards embryo research.

When the technology is ready, even more provocative reproductive feats might be possible. For instance, cells from a man’s skin could theoretically be used to create eggs that are fertilized with a partner’s sperm, then nurtured in the womb of a surrogate. Some doubt, however, that such a feat would ever be possible — the Hinxton Group, an international consortium of scientists that discusses stem-cell ethics and challenges, concluded that it would be difficult to get eggs from male XY cells and sperm from female XX cells. “The instructions that the female niche is supplying to the male cell do not coordinate with each other,” says Clark, a member of the consortium.

Saitou used iPS cells from male mice to create sperm and from female mice to create eggs, but he says that the reverse should be possible. If so, eggs and sperm from the same mouse could be generated and used for fertilization, producing something never seen before: a mouse created by self-fertilization. Neither Hayashi nor Saitou is ready to try this. “We would only do this [in mice] if there were a good scientific reason,” says Saitou. Right now he does not see one.

The two scientists already feel some pressure from patients and Japanese funding agencies to move forward. The technique could be a last hope for women who have had no luck with IVF, or for people who had cancer in childhood and have lost the ability to produce sperm or eggs. Hayashi warns those who write to him that a viable infertility treatment could be 10 or even 50 years in the future. “My impression is that it is very far away. I don’t want to give people unfeasible hope,” he says.

Patients see the end result — success in mice — and often ignore the years of painstaking work that led to such a technical tour de force. They do not realize that switching from mice to humans means starting again almost from scratch, says Hayashi. The human early embryo is so different from the mouse that it is almost “like starting over on a process that took more than ten years”.

Source: Nature. 

 

 

 

Japan to offer fast-track approval path for stem cell therapies.


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 A retooling of Japan’s drug authorization framework, on its way to becoming law, could produce the world’s fastest approval process specifically designed for regenerative medicine. “I don’t know of any other countries that have broken out with a separate and novel system” for cellular therapies, says University College London regenerative medicine expert Chris Mason, who recently met with Japanese policymakers to discuss the law.

Japan has recently been trying to shake its ‘drug lag’, a term used to describe its historically slow review process that sometimes translates into therapies reaching the market well after they have received the green light elsewhere. But the country is now ready to speed the translation of regenerative medicine to the bedside.

The move comes in response to the potential offered by its homegrown induced pluripotent stem (iPS) cell technology, which netted Shinya Yamanaka, of the University of Kyoto, last year’s Nobel Prize in Medicine or Physiology. The government already flooded the field with more than 20 billion yen ($206 million) in a supplementary budget announced earlier this year, and it’s expected to allocate another 90 billion yen into the sector over the coming decade.

Under the Pharmaceutical Affairs Law as it currently stands, regenerative therapies, like small-molecule drugs, must undergo three phases of costly and cumbersome clinical trials to get approval by Japan’s Pharmaceutical and Medical Devices Agency.

The proposed amendments to the pharmaceutical law will create a new, separate approval channel for regenerative medicine. Rather than using phased clinical trials, companies will have to demonstrate efficacy in pilot studies of as few as ten patients in one study, if the change is dramatic enough, or a few hundred when improvement is more marginal. According to Toshio Miyata, deputy director of the Evaluation and Licensing Division at the Pharmaceutical and Food Safety Bureau in Tokyo, if efficacy can be “surmised,” the treatment will be approved for marketing. At that stage, the treatment could be approved for commercial use and, crucially for such expensive treatments, for national insurance coverage.

Phased out

With the bar for regenerative therapies dramatically lowered by requiring only limited safety and efficacy data—and essentially doing away with the need for high-powered phase 3 trials—the amendments’ architects say it will be possible to get a stem cell treatment to the market in just three years, rather than the typical six or more. The law should also give local producers of regenerative medicine an edge even over those selling stem cell therapies in South Korea, where an accelerated system has helped companies get more stem cell treatments on the market than any other country (see Nat. Med. 18, 329, 2012). “It’s bold,” says Yoshihide Esaki, director of Bio-Industry Division, a bureau of the Ministry of Economy, Trade and Industry based in Tokyo, which promoted legislation calling for the update.

Following approval, there will be a post-market surveillance period of five to seven years, after which the treatment will be evaluated again for safety and efficacy. Every patient must be entered in a registry during that period, says Miyata. If the therapies prove inefficacious or unsafe, approval can be withdrawn.

Doug Sipp worries whether post-market surveillance will turn up relevant data. Sipp, who studies regulatory issues related to stem cells at the RIKEN Center for Developmental Biology in Kobe, Japan, says that making people who receive the therapies during this period cough up even the 30% co-pay generally required under Japan’s national insurance plan “will essentially be asking patients to pay for the privilege of serving as the subjects of medical experiments.” And since the patients are paying, the studies cannot be randomized or blinded. Paying patients are also more likely to experience placebo effects, Sipp warns.

“There’s also the opportunity costs to patients,” who might be able to find better therapies elsewhere, adds Mason. “We have to make sure these therapies are safe and effective. Otherwise these regulatory routes are going to be closed.”

Despite these concerns, passage of the pre-vetted law is almost a given. Esaki says there’s a 50% chance the Japanese parliament will pass the law during the current session, ending in June. If so, it would go into effect next April. If not, scientists might have to wait until November 2014 or as late as April 2015.

Source: Nature

Life created from eggs made from skin cells.


Stem cells made from skin have become “grandparents” after generations of life were created in experiments by scientists in Japan.

The cells were used to create eggs, which were fertilised to produce baby mice. These later had their own babies.

If the technique could be adapted for people, it could help infertile couples have children and even allow women to overcome the menopause.

But experts say many scientific and ethical hurdles must be overcome.

Healthy and fertile

Stem cells are able to become any other type of cell in the body from blood to bone, nerves to skin.

Last year the team at Kyoto University managed to make viable sperm from stem cells. Now they have performed a similar feat with eggs.

They used stem cells from two sources: those collected from an embryo and skin-like cells which were reprogrammed into becoming stem cells.

The first step, reported in the journal Science, was to turn the stem cells into early versions of eggs.

A “reconstituted ovary” was then built by surrounding the early eggs with other types of supporting cells which are normally found in an ovary. This was transplanted into female mice.

Surrounding the eggs in this environment helped them to mature.

IVF techniques were used to collect the eggs, fertilise them with sperm from a male mouse and implant the fertilised egg into a surrogate mother.

Dr Katsuhiko Hayashi, from Kyoto University, told the BBC: “They develop to be healthy and fertile offspring.”

Those babies then had babies of their own, whose “grandmother” was a cell in a laboratory dish.

Devastating blow

The ultimate aim of the research is to help infertile couples have children. If the same methods could be used in people then cells in skin could be turned into an egg. Any resulting child would be genetically related to the mother.

However, Dr Hayashi said that was still a distant prospect: “I must say that it is impossible to adapt immediately this system to human stem cells, due to a number of not only scientific reasons, but also ethical reasons.”

He said that the level of understanding of human egg development was still too limited. There would also be questions about the long-term consequences on the health of any resulting child.

Dr Evelyn Telfer, from the University of Edinburgh, said: “It’s an absolutely brilliant paper – they made oocytes [eggs] from scratch and get live offspring. I just thought wow! The science is quite brilliant.”

However, she warned that this had “no clinical relevance” as there were still too many gaps in understanding about how human eggs developed.

“If you can show it works in human cells it is like the Holy Grail of reproductive biology,” she added.

Prof Robert Norman, from the University of Adelaide, said: “For many infertile couples, finding they have no sperm or eggs is a devastating blow.

“This paper offers light to those who want a child, who is genetically related to them, by using personalised stem cells to create eggs that can produce an offspring that appears to be healthy.

“It also offers the potential for women to have their own children well past menopause raising even more ethical issues.

“Application to humans is still a long way off, but for the first time the goal appears to be in sight.”

Dr Allan Pacey, from the British Fertility Society and the University of Sheffield, said: “What is remarkable about this work is the fact that, although the process is still quite inefficient, the offspring appeared healthy and were themselves fertile as adults.”

Source:BBC

 

 

Human Brains Outpace Chimp Brains in Womb.


Humans‘ superior brain size in comparison to their chimpanzee cousins traces all the way back to the womb. That’s according to a study reported in the September 25 issue of Current Biology, a Cell Press publication, that is the first to track and compare brain growth in chimpanzee and human fetuses.

“Nobody knew how early these differences between human and chimp brains emerged,” said Satoshi Hirata of Kyoto University.

Hirata and colleagues Tomoko Sakai and Hideko Takeshita now find that human and chimp brains begin to show remarkable differences very early in life. In both primate species, the brain grows increasingly fast in the womb initially. After 22 weeks of gestation, brain growth in chimpanzees starts to level off, while that of humans continues to accelerate for another two months or more. (Human gestation time is only slightly longer than that of chimpanzees, 38 weeks versus 33 or 34 weeks.)

The findings are based on 3D ultrasound imaging of two pregnant chimpanzees from approximately 14 to 34 weeks of gestation and comparison of those fetal images to those of human fetuses. While early brain differences were suspected, no one had previously measured the volume of chimpanzee brains as they develop in the womb until now.

The findings are part of a larger effort by the research team to explore differences in primate brains. In another Current Biology report published last year, they compared brain development in chimps versus humans via magnetic resonance imaging (MRI) scans of three growing chimpanzees from the age of six months to six years.

“Elucidating these differences in the developmental patterns of brain structure between humans and great apes will provide important clues to understand the remarkable enlargement of the modern human brain and humans’ sophisticated behavior,” Sakai said.

The researchers say they now hope to explore fetal development in particular parts of the brain, including the forebrain, which is critical for decision making, self-awareness, and creativity.

Source: http://www.sciencedaily.com

Stem-cell pioneer banks on future therapies.


Japanese researcher plans cache of induced stem cells to supply clinical trials.

Progress toward stem-cell therapies has been frustratingly slow, delayed by research challenges, ethical and legal barriers and corporate jitters. Now, stem-cell pioneer Shinya Yamanaka of Kyoto University in Japan plans to jump-start the field by building up a bank of stem cells for therapeutic use. The bank would store dozens of lines of induced pluripotent stem (iPS) cells, putting Japan in an unfamiliar position: at the forefront of efforts to introduce a pioneering biomedical technology.

A long-held dream of Yamanaka’s, the iPS Cell Stock project received a boost last month, when a Japanese health-ministry committee decided to allow the creation of cell lines from the thousands of samples of fetal umbilical-cord blood held around the country. Yamanaka’s plan to store the cells for use in medicine is “a bold move”, says George Daley, a stem-cell biologist at Harvard Medical School in Boston, Massachusetts. But some researchers question whether iPS cells are ready for the clinic.

Yamanaka was the first researcher to show, in 2006, that mature mouse skin cells could be prodded into reverting to stem cells1 capable of forming all bodily tissues. The experiment, which he repeated2 with human cells in 2007, could bypass ethical issues associated with stem cells derived from embryos, and the cells could be tailor-made to match each patient, thereby avoiding rejection by the immune system.

Japan is pumping tens of millions of dollars every year into eight long-term projects to translate iPS cell therapies to the clinic, including a US$2.5-million-per-year effort to relieve Parkinson’s disease at Kyoto University’s Center for iPS Cell Research and Application (CiRA), which Yamanaka directs. That programme is at least three years away from clinical trials. The first human clinical trials using iPS cells, an effort to repair diseased retinas, are planned for next year at the RIKEN Center for Developmental Biology in Kobe.

Those trials will not use cells from Yamanaka’s Stock. But if they or any other iPS cell trials succeed, demand for the cells will explode, creating a supply challenge. Deriving and testing iPS cells tailored to individual patients could take six months for each cell line and cost tens of thousands of dollars.

Yamanaka’s plan is to create, by 2020, a standard array of 75 iPS cell lines that are a good enough match to be tolerated by 80% of the population. To do that, Yamanaka needs to find donors who have two identical copies of each of three key genes that code for immune-related cell-surface proteins called human leukocyte antigens (HLAs). He calculates that he will have to sift through samples from some 64,000 people to find 75 suitable donors.

Using blood from Japan’s eight cord-blood banks will make that easier. The banks hold some 29,000 samples, all HLA-characterized, and Yamanaka is negotiating to gain access to those that prove unusable for other medical procedures. One issue remains unresolved: whether the banks need to seek further informed consent from donors, most of whom gave the blood under the understanding that it would be used for treating or studying leukaemia. Each bank will determine for itself whether further consent is needed.

Yamanaka has already built a cell-processing facility on the second floor of CiRA and is now applying for ethics approval from Kyoto University to create the stock. Takafumi Kimura, a CiRA biologist and head of the project’s HLA analysis unit, says that the team hopes to derive the first line, carrying a set of HLA proteins that matches that of 8% of Japan’s population, by next March.

Yamanaka’s project has an advantage in that genetic diversity in Japan is relatively low; elsewhere, therapeutic banks would have to be larger and costlier. Most iPS banks outside Japan specialize in cells from people with diseases, for use in research rather than treatment. The California Institute for Regenerative Medicine (CIRM) in San Francisco, for example, plans to bank some 3,000 cell lines for distribution to researchers.

Alan Trounson, president of CIRM, says that unresolved research questions about iPS cells make it “premature” to begin therapeutic trials. “We don’t have complete pictures of how good they would be,” he says, noting that such cells accumulate mutations and other defects as they are produced from differentiated cells. Irving Weissman, a stem-cell biologist at Stanford University in California, warns that iPS cells derived from blood cells have been shown to form tumours3.

Kimura says that the answer is to carefully avoid the white blood cells that cause tumours when deriving the cell lines, and he stresses that all safety concerns will be addressed. “We’re building a national resource. It has to be safe and have the confidence of the people.”

Daley, who last month toured CiRA’s facility, calls it “nothing short of spectacular, pristine, perfect”. He agrees that proving the safety of the cells will be tough, but he is enthusiastic about the effort. “It’s clear they’re readying themselves for a big project,” he says.

Source: Nature.