stem cells

Pigs pave the way for advancements in IVF treatment

New research makes IVF four times more efficient to create pigs like this for genetics research and breeding in labs like that of Randy Prather at MU. | Photo by Nicholas Benner.

New research makes IVF four times more efficient to create pigs like this for genetics research and breeding in labs like that of Randy Prather at MU. | Photo by Nicholas Benner.

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.

Their discovery doubles the number of piglets born and speeds up the entire IVF process by 400 percent, which significantly increases both the efficiency of experiments and their potential application to other species. The journal Proceedings of the National Academy of Sciences published their work July 3 in its online early edition.

 

From the beginning:

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.

Jacqueline Ihnat #IAmScience

Jacqueline Ihnat

Jacqueline Ihnat, one of the 12 Cherg Summer Scholars chosen from within the Honors College at MU in 2017. | Photo by Mary Jane Rogers, Bond LSC

By Mary Jane Rogers | Bond LSC

“#IAmScience because I am able to apply what I learn in the classroom to research that makes progress towards a better future.”

Jacqueline Ihnat was recently selected as one of the 12 Cherng Summer Scholars within the Honors College at the University of Missouri. This scholarship provides her with $8,000 to fund her summer research. She’s fascinated with cells and how our bodies function, so this summer she’s studying the role of specialized stem cells in muscle regeneration and how they interact with muscle fibers—specifically the role of Eph-A3, a type of cell-surface receptor. Her faculty mentor for this project is Dawn Cornelison, an investigator in MU’s Bond Life Sciences Center.

“Doing research helps keep me focused on the bigger picture,” Ihnat said.

“Sometimes in class we learn things that don’t seem entirely relevant or useful, but being part of a research lab allows me to apply some of the knowledge that I gain in the classroom. It’s a daily reminder of why I’m learning what I am.”

How does Zika move from mother to child?

Scientists use placental cells in lab to study virus
By Phillip Sitter | MU Bond Life Sciences Center

DSC_1893.jpg

Megan Sheridan, an MU grad student, removes the base solution from a demonstrated sample of stem cells that will be grown into placental cells for study of Zika virus. Within four days of exposure to the correct hormones, the stem cells express genes of placental cells, and within another day start producing placental hormones. The cells are infected with Zika at day four to ensure maximum measurable interaction, as the stem cells naturally die in culture after about ten days. | photo by Phillip Sitter, Bond LSC

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.

DSC_1899.jpg

Megan Sheridan sits in front of a demonstration of her work with pluripotent stem cells. Sheridan is a graduate student who works in Toshihiko Ezashi’s lab, where she produces cells with placental characteristics from the stem cells in order to study placenta interaction with Zika virus. | photo by Phillip Sitter, Bond LSC

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.