Roberts honored for breakthrough discovery in reproductive biology 30 years ago
By Eleanor C. Hasenbeck | Bond Life Sciences Center
In 1987, Michael Roberts published a groundbreaking discovery that changed the world of reproductive biology research.
Roberts and members of his lab discovered that a type of protein, an interferon, impacted how the bodies of animals such as sheep, goats and cows, recognized an embryo early in pregnancy. Previously thought to only be a part of a cell’s immune system response, this new signaling role changed the field.
In honor of his lab’s groundbreaking discovery, Roberts recently curated a section of six reviews examining the history of the discovery and current research that has built on it for the November issue of the journal Reproduction.
The discovery revealed an unknown in the reproductive systems of the ruminant family of animals, including sheep, goats, cows and deer. When an embryo first begins developing, before it’s placenta even attaches to the uterus, it releases interferons. Only present for a few days, these proteins signal to the mother’s body that the embryo is there. It triggers the response that keeps the animal from going into heat, basically shifting the animal’s hormones from breeding mode to pregnancy mode.
If the embryo doesn’t release interferons, the mother miscarries. Placing interferons in sheep that were not pregnant made the animals pseudopregnant, a false pregnancy in which no fetus is present.
Scientists at the time knew something made the mother’s body recognize the embryo, but they were not sure what. The discovery of interferon-tau was a mystery solved. That this ‘something’ was an interferon was also a surprise. Before Roberts and his co-discoverer, Fuller Bazer, found interferon-tau, researchers thought that interferons only function was in the immune system. Other interferons help the body recover from viral infections, like cold and influenza, Roberts said. The discovery that the protein also played a role in pregnancy caused some hubbub. It even caught the attention of The New York Times, Roberts said.
“It opened up a whole new area,” he said. “We all the sudden understood how these animals got pregnant, so people went off in all sorts of directions with it.”
The discovery of interferon-tau created opportunities for more research in how ruminant’s unique reproductive systems evolved. Other studies focused on using interferon-tau to improve livestock fertility, but ultimately this interest fizzled out as researchers found fertility treatments for cows were cost-ineffective for producers and unappealing to the public.
The discovery of interferon-tau earned Roberts and his co-discoverer the Wolf Prize in agriculture in 2002. Some consider the prize an equivalent to the Nobel Prize since the Nobel prize does not regularly honor agriculturalists.
After the discovery of interferon tau, Roberts found another protein that impacts pregnancy, which formed the basis of a pregnancy test for cows. Roberts said it’s now a multi-million dollar product in the cattle industry.
Today, Roberts’ lab has moved to other developmental research. He started studying human placentas. His work focuses on preeclampsia, a condition which impacts 5-10 percent of all pregnancies and is caused by the placenta. Roberts’s lab has also developed new lines of pluripotent pig stem cells which are helping scientists learn how to regenerate eye and heart tissue. At age 77, he is still funded and active.
“#IAmScience because it’s extraordinary knowing that a small step towards a treatment could positively impact someone’s life down the road.”
Megan Sheridan doesn’t let anything slow her down.
From presenting at the Society for the Study of Reproduction’s Trainee Research Competition last week—and winning first place—to finishing up her thesis while working in Dr. Michael Roberts’ lab, she’s always juggling multiple projects. Sheridan is finishing up a Ph.D in biochemistry and hopes to graduate in December 2017 or May 2018, depending on how quickly she finishes writing her thesis.
“I was lucky enough to pick up a project studying Zika virus infections early in pregnancy,” she said. “It was one of those perfect timing moments, and we ended up getting some pretty exciting results off the bat. Now I’m really inspired by the direction my thesis work is going and find that my projects are very different but that makes things exciting.“
Sheridan’s thesis focuses on using stem cells as a model for early placenta development and how preeclampsia and viral infections like Zika impact a pregnancy. Preeclampsia is a condition during pregnancy that causes high blood pressure and protein in the urine. The disease likely occurs in the first trimester, but the symptoms don’t evolve until the 2nd or 3rd trimester. To study it, Sheridan uses stem cells generated from umbilical cords of babies born to mothers experiencing preeclampsia or a normal pregnancy, and then uses those cells to determine what defects in the placenta might contribute to the disease preeclampsia.
“I would like to learn as much as possible about the placenta and human pregnancy,” she said. “There are so many unknowns in this area of research because you can’t access the placenta during a pregnancy without disrupting the pregnancy. There are many complications that effect the mother and baby, and if more was known about normal placenta development in pregnancy, then we may be able to better understand and prevent some of those complications.”
Sheridan completed her undergraduate degree at MU, and urges undergraduates to get started in research early, as she believes it gives students a stronger foundation for graduate school. She also believes that mistakes are part of the research process, and wasn’t afraid to share one that she made early on in the Ph. D program.
“I remember in my very first rotation as a graduate student I was learning how to transfect cells with DNA so we could do a reporter assay. We were in the process of adding all the reagents, and between the student I was working with and myself we got confused about who added what,” she laughed. “Somehow, we never added the DNA- an integral part of the transfection! So a week later when we were analyzing the data, we noticed there were no values at all.”
After graduation, Sheridan hopes to experience living outside of Missouri for her postdoc placement. She’d like to stay in academia, and looks forward to continuing to research and teach.
Perhaps one day she’ll even return to MU and Bond LSC!
Research quadruples speed and efficiency to develop embryos
By Samantha Kummerer | Bond LSC
What started as a serendipitous discovery is now opening the door for decreasing the costs and risks involved with in vitro fertilization (IVF).
And it all started with cultured pig cells.
Dr. Michael Roberts’ and Dr. Randall Prather’s laboratories in the University of Missouri work with pigs to research stem cells. During an attempt to improve how they grew these cells, researchers stumbled across a method to improve the success of IVF in pigs.
“Sometimes you start an experiment and come up with up with a side project and it turns out to be really good,” Researcher Ye Yuan said.
The Prather lab in the MU Animal Sciences Research Center uses genetically modified pig embryos to improve pig production for agriculture and also to mimic human disease states, such as cystic fibrosis. Roberts’ team in the Bond Life Sciences Center occasionally collaborates with Prather’s lab to produce genetically modified pigs for this valuable research. However, the efficiency of producing these pigs is very low because it depends on multiple steps.
First, scientists remove oocytes (“eggs”) and the “nurse” cells that surround them from immature female pig ovaries and place the eggs in a chemical environment designed to mature the eggs, allowing them to be fertilized in vitro with sperm from a boar. This process creates zygotes, which are single-celled embryos, that are allowed to develop further until they become hollow balls of cells called blastocysts about six-days later. These tiny embryos are then transferred back into a female pig with the hopes of achieving a successful pregnancy and healthy piglets.
However, Roberts said the chance of generating a successful piglet after all those steps is very low; only 1-2 percent of the original eggs make it that far.
The quality of the premature eggs and the process of maturing them significantly reduces the rate of success.
“In other words, all this depends on having oocytes that are competent, that is they can be fertilized, form blastocysts and initiate a successful pregnancy,” Roberts explained.
Normally, researchers overcome the low success rate by starting out with a very large number of eggs, but this takes lots of time and money.
So, lab researchers, Ye Yuan and Lee Spate, began tinkering with the way the eggs were cultured before they were fertilized, making use of special growth factors they used when culturing pig embryonic stem cells.
Yuan and Spate added two factors called fibroblast growth factor 2 (FGF2) and leukemia inhibitory factor (LIF).
This combination helped more than the use of just a single factor and so they decided to add a third factor, insulin-like growth factor 1 (IGF1).
Together the three compounds create the chemical medium termed “FLI”.
“It improved every aspect of the whole process,” Roberts said. “It almost doubled the efficiency of oocyte maturation in terms of going through meiosis. It appeared to improve fertilization and it improved the production of blastocysts.”
In all, the use of FLI medium doubles the number of piglets born and quadruples the efficiency of the entire process from egg to piglet.
While the researchers are still figuring out why the three factors work together so well, Roberts believes it has to do with the fluid that surrounds the immature eggs while they are still in the ovary.
Roberts explained that unusual metabolic changes happen in the eggs and their nurse cells when the three components are used in combination but not when they are used on their own. These components are also found in the follicular fluid surrounding the egg when it is in the ovary.
However, follicular fluid actually contains factors that hinder egg maturation until the time is right, so it would seem counterintuitive to add the fluid to a chemical environment aimed at maturing the eggs. However, when freed from the other components of follicular fluid, the three growth factors act efficiently to promote maturation.
“It just creates this whole nurse environment for that egg. Once you’ve done that you’ve sort of patterned them to do everything else after that properly — fertilization, development of that fertilized egg to form a blastocyst, and the capability of those blastocysts to give rise to a piglet,” Roberts said.
Researchers hope the FLI medium can be translated beyond genetically modified pigs.
“If we could translate this to other species it could be more meaningful,” Yuan explained.
For the cattle industry, FLI has the potential to decrease the time between generations in highly prized animals.
Currently, if an immature dairy cow has desirable traits, the industry has to wait a year or so for that cow to mature and for its eggs to be collected. Using FLI medium immature eggs could be retrieved when the prized female is still a calf. After fertilizing them with semen from a prized bull, production of more cows with desirable traits could be achieved in a shorter amount of time.
The potential implications of this discovery aren’t just for farm animals.
Yuan said if this treatment could be applied to humans it would be a big help for both the patient and the whole field of human IVF.
Currently, in vitro fertilization for humans comes with high costs and risks.
“You try to generate a lot of eggs from the patients by using super-high doses of expensive hormones, which is not necessarily good for the patient and can, in fact, be risky. ” Roberts explained.
These eggs are then collected, fertilized, and the best-looking embryo transferred back to the patient. As in pigs, this overall process isn’t all that efficient. The hope is that the treatment of the patient with hormones can be minimized if immature eggs are collected directly from the ovary by using an endoscope and matured in FLI medium, allowing them to be just as competent as those retrieved after high hormone treatment.
“The idea is it would be safer for the woman, it would be cheaper, and it might even achieve a better success rate,” Roberts said.
The team still has some time before knowing for sure if FLI medium is applicable in other mammals.
Yuan said the focus is now on understanding the mechanism behind how the three compounds work so well together.
For now, preliminary data are being collected with mice and a patent is awaiting approval. Still, the team has high hopes for this almost accidental finding.
“Whenever you’re doing science, you’d like to think you’re doing something that could be useful,” Roberts said. “I mean when we started this out it wasn’t to improve fertility IVF in women, it was to just get better oocytes in pigs. Now it’s possible that FLI medium could become important in bovine embryo work and possibly even help with human IVF.”
Michael Roberts is a Bond LSC scientist and a Curators’ Distinguished Professor of Animal Science, Biochemistry and Veterinary Pathobiology in the College of Agriculture, Food and Natural Resources (CAFNR) and the College of Veterinary Medicine. He is also a member of the National Academy of Sciences.
Randall Prather is a Curators’ Distinguished Professor of Animal Science in the College of Agriculture, Food and Natural Resources (CAFNR) and Director of the National Institutes of Health funded National Swine Resource and Research Center.
What if you could have pork without the pig? Nicholas Genovese’s cultured meat could provide a more environmentally friendly meat
By Eleanor C. Hasenbeck | MU Bond Life Sciences Center
Scientists are one step closer to that reality. For the first time, researchers in the Roberts’ lab at Bond Life Sciences Center at MU were able to create a framework to make pig skeletal muscle cells from cell cultures.
In vitro meat, also known as cultured meat or cell-cultured meat, is made up of muscle cells created from cultured stem cells.
As a visiting scholar at the University of Missouri, Nicholas Genovese mapped out pathways to successfully create the first batch of in vitro pork. Genovese also said it was the first time it was done without an animal serum, a growth agent made from animal blood.
According to Genovese, his research in the Roberts Lab was also the first time the field of in vitro meats was studied at an American university.
“I feel it’s a very meaningful way to create more environmentally sustainable meats, which is going to use fewer resources, with fewer environmental impacts and reduce need for animal suffering and slaughter while providing meats for everyone who loves meat,” Genovese said.
The research could have environmental impacts. According to the United Nation’s Food and Agriculture Organization, livestock produce 14.5 percent of all human-produced greenhouse gas emissions. Livestock grazing and feed production takes up 59 percent of the earth’s un-iced landscape. Cultured meat takes up only as much land as the laboratory or kitchen (or carnery, the term some members of the industry have coined for their facilities) it is produced in. It uses energy more efficiently. According to Genovese, three calories of energy can produce one calorie of consumable meat. The conversion factor in meat produced by an animal is much higher. According to the FAO, a cow must consume 11 calories to produce one calorie of beef for human consumption.
And while Michael Roberts, the lab’s principal investigator, is skeptical of how successful in vitro meat will be, he said the results could yield other benefits. Researchers might be able to use a similar technique as they used to create skeletal muscle tissue to make cardiac muscle tissue. Pork muscles are anatomically similar to a human’s and can be used to model treatments for regenerative muscle therapies, like replacing tissue damaged by injury or heart attacks.
“I was interested in using these cells to show that we could differentiate them into a tissue. It’d been done with human and mouse, but we’re not going to eat human and mouse,” Roberts said. “The pig is so similar in many respects to humans, that if you’re going to test out technology and regenerative medicine, the pig is really an ideal animal for doing this, particularly for heart muscle,” he added.
Scientists use placental cells in lab to study virus
By Phillip Sitter | MU Bond Life Sciences Center
Scientists believe they have a better way to study how Zika virus can spread from a pregnant mother to her fetus — and their technique doesn’t even involve observations of babies in the womb or post-natal examinations.
“As soon as we heard about Zika, everybody’s light bulbs turned on,” said Megan Sheridan, a graduate student at the University of Missouri Bond Life Sciences Center.
Sheridan works in the lab of Toshihiko Ezashi at Bond LSC, and she, in turn, is part of a cross-campus team researching Zika with R. Michael Roberts, Alexander Franz, Danny Schust and Ezashi.
Roberts’ lab studies pluripotent stem cells — progenitor cells which can develop into any other type of cell in the body.
“We use the proper signals to drive stem cells to become like placental cells,” Sheridan explained. With this capability to stimulate stem cells with growth hormones and inhibitors at opportune moments, Roberts’ researchers realized they could create enough placental cells to create an environment similar to that of a womb in very early stages of pregnancy.
This is something which Sheridan thinks hasn’t been done before in regards to studying placental interaction with Zika. Their technique could give a look into the first trimester, when epidemiological studies say a fetus is most susceptible to infection.
Roberts’ lab is trying to understand the placental barrier’s vulnerability to Zika virus in its early stage of pregnancy. During this time, an infection could occur even before the mother is aware she is pregnant.
If the lab uses their technique to understand how Zika virus enters placental cells, then potentially they could also learn how to strengthen the placenta as a barrier to Zika and make it a first line of defense against infection of the fetus in the womb. If developing babies don’t get infected with Zika, then they won’t suffer the consequences of birth defects.
One such defect is microcephaly where a baby is born with a smaller than expected head, which may in turn be a sign that their brain has not fully developed. While infection with Zika virus is rarely fatal or otherwise severe in itself — many people don’t even develop symptoms — birth defects like microcephaly could cause further developmental problems like delays in learning how to speak and walk, intellectual disabilities, difficulty swallowing and problems with hearing and vision, according to global health organizations.
Microcephaly only became a widely documented effect of Zika after a particular strain surged across South and Central America with the infected mosquitoes that carry it, Sheridan explained, but this may be in part because previous Zika infections and outbreaks were themselves poorly documented.
While birth defects caused by Zika have drawn much media attention as the disease has spread northward through our hemisphere from Brazil, studies focusing on infection in the womb have only used placental material that has come to term. This may not be the most accurate way to see how the placenta gets infected in the first place early in pregnancy.
The pathway of Zika virus infection in lab mice isn’t really comparable to human infection, because mice aren’t infected with this virus naturally. Only lab mice that have had their genomes altered to be able to acquire the virus have susceptibility to the infection that can be modeled.
Roberts’ lab is currently working with the African strain of Zika and obtained strains from Southeast Asia and Central America recently. There’s about a 99 percent genetic similarity across strains, Sheridan said.
Zika virus was first discovered in Africa in Uganda in 1947, according to the Centers for Disease Control and Prevention. The first human case was documented in 1952, and subsequent outbreaks also occurred in Southeast Asia and the Pacific Islands. The Pan American Health Organization issued an alert about the confirmed arrival of the virus in Brazil in May 2015.
The lab has completed Zika infections of some of their stem cell-produced placental cells. Sheridan reassured that even though the lab works with live viruses, Zika is not airborne, and none of their work involves mosquitoes.
Roberts’ lab submitted one grant application earlier this year to the National Institutes of Health for funding for their research. While that application was denied, Sheridan said that they have a lot more preliminary data now and are hoping to submit a revised grant soon.
She said that their original work was “highly scored, but the funding level is still low,” meaning that obtaining funds for research into Zika virus is highly competitive nationally.
Legislation to fund more efforts into studying and preventing transmission of Zika virus is caught in congressional gridlock, according to The New York Times and other media outlets.
In the mean time, as the Roberts lab prepares its next grant application submission, Sheridan said of her efforts that she is “working hard to make progress on the project as quickly as possible.”
Please visit the CDC’s dedicated page for more information on Zika virus — including advice for travellers and pregnant women, description of symptoms and treatment, steps you can take to control mosquitoes and prevent other means of transmission of the virus and more background on the history and effects of the disease.
New line of pigs do not reject transplants, will allow for future research on stem cell therapies
Story by Nathan Hurst/MU News Bureau
COLUMBIA, Mo. – One of the biggest challenges for medical researchers studying the effectiveness of stem cell therapies is that transplants or grafts of cells are often rejected by the hosts. This rejection can render experiments useless, making research into potentially life-saving treatments a long and difficult process. Now, researchers at the University of Missouri have shown that a new line of genetically modified pigs will host transplanted cells without the risk of rejection.
“The rejection of transplants and grafts by host bodies is a huge hurdle for medical researchers,” said R. Michael Roberts, Curators Professor of Animal Science and Biochemistry and a researcher in the Bond Life Sciences Center. “By establishing that these pigs will support transplants without the fear of rejection, we can move stem cell therapy research forward at a quicker pace.”
In a published study, the team of researchers implanted human pluripotent stem cells in a special line of pigs developed by Randall Prather, an MU Curators Professor of reproductive physiology. Prather specifically created the pigs with immune systems that allow the pigs to accept all transplants or grafts without rejection. Once the scientists implanted the cells, the pigs did not reject the stem cells and the cells thrived. Prather says achieving this success with pigs is notable because pigs are much closer to humans than many other test animals.
“Many medical researchers prefer conducting studies with pigs because they are more anatomically similar to humans than other animals, such as mice and rats,” Prather said. “Physically, pigs are much closer to the size and scale of humans than other animals, and they respond to health threats similarly. This means that research in pigs is more likely to have results similar to those in humans for many different tests and treatments.”
“Now that we know that human stem cells can thrive in these pigs, a door has been opened for new and exciting research by scientists around the world,” Roberts said. “Hopefully this means that we are one step closer to therapies and treatments for a number of debilitating human diseases.”
Roberts and Prather published their study, “Engraftment of human iPS cells and allogeneic porcine cells into pigs with inactivated RAG2 and accompanying severe combined immunodeficiency” in the Proceedings of the National Academy of Sciences.
This study was made possible through grants from Konkuk University in South Korea and the National Institutes of Health.
Roberts has appointments in the MU College of Food, Agriculture and Natural Resources (CAFNR) and the MU School of Medicine and is a member of the National Academy of Sciences. Prather has an appointment in CAFNR and is the director of the NIH-funded National Swine Resource and Research Center.
You can’t see the resemblance, but cells in Michael Roberts’ lab share a family tree with some newborns.
Their common genetics may help explain severe, early-onset preeclampsia, an inherited disorder that leads to a placenta that is often small and inefficient and possibly due to the mother’s body not fully welcoming her pregnancy.
University of Missouri Health Center scientists such as Danny Schust and Laura Schulz, work with Roberts and Toshihiko Ezashi, both Bond Life Sciences Center reproductive biologists, to search for its complex cause.
That starts in the delivery room. OBGYN’s Danny Schust and his residents save small pieces of umbilical cord from preeclampsia pregnancies, allowing Roberts and Ezashi to grow cells with the disease.
“We’re essentially recreating the previous pregnancy, going back in time as far as that baby is concerned,” Roberts said. “That allows us to look at the disease in a Petri dish, look at the properties of these cells to try and figure out what’s wrong with them.”
Preeclampsia affects 3-7 percent of births worldwide, and leads to around 50,000 deaths annually. Symptoms like high blood pressure and protein in the urine tip off doctors to the disorder, but it can go unnoticed until late in the pregnancy unless the symptoms are severe. Left unchecked the disease can lead to seizures and death. The only cure for the serious early-onset form of the disease is to deliver the baby prematurely, usually between 28-33 weeks. This leaves the newborn underweight and with complications like underdeveloped lungs.
Creating useful cells from the collected umbilical cords takes a little bit of work. Cells grown from the umbilical cords are converted into induced pluripotent stem cells, with potential to become any type of cell in the body. Researchers then use a series of hormones, growth factors and other conditions to create placental cells mirroring the previous pregnancy.
Normally the placenta – containing genes from both mother and father – grows into the wall of the uterus to establish a supply of blood, nutrients and oxygen to support the embryo. But in preeclampsia, those cells encounter problems.
Roberts and Schulz focus on extravillous trophoblasts, placental cells that invade the wall of the womb, while collaborators Toshihiko Ezashi and Danny Schust study synctiotrophoblasts, placental cells responsible for uptake of oxygen and nutrients from the mother’s blood.
“The question becomes do these placental cells grow too much or too little, and it appears that in preeclampsia they don’t grow enough,” Roberts said. “We’re just beginning to look at their ability to move and grow through a jelly-like substance that impedes a cell’s mobility and ability to pass through small pores on a membrane. We’re also comparing the gene expression of the cells from preeclamptic patients with those from normal births..”
The Roberts/Ezashi/Schulz/Schust team still has several years left on two five-year National Institutes of Health grants and hopes to narrow the search for genes linked to preeclampsia. If they pinpoint the culprit genes, scientists could one day potentially correct the problem by developing drugs that restore normality to the placental cells or even by using an induced pluripotent stem cell approach.
“It might mean you could go back to correct the defect in those cells or take that patient’s cells, make some normal cells and perhaps substitute them back in to effect a cure,” Roberts said. “My own feelings are that we’re a very long way from doing that, but that is the thought.”
Michael Roberts is a Curators’ Professor of Animal Science, Biochemistry and Veterinary Pathobiology in the College of Agriculture, Food and Natural Resources (CAFNR) and the College of Veterinary Medicine, respectively. Toshihiko Ezashi is a research associate professor in CAFNR. Danny Schust is an associate professor and Laura Schulz an assistant professorof Obstetrics, Gynecology, & Women’s Health, at MU’s School of Medicine.