Induced cardiac progenitor cells (iCPCs) can develop in a dish into contracting heart muscle cells (green) when grown together with other contracting heart muscle cells. These cells could potentially be used for modeling heart diseases as well as testing drugs. Video: Pratik Lalit
By genetically reprogramming the most common type of cell in mammalian connective tissue, researchers at the University of Wisconsin-Madison have generated master heart cells — primitive progenitors that form the developing heart.
Timothy Kamp, leader of the team that transformed mouse fibroblasts into primitive master heart cells. Photo: Jeff Miller
Writing online Feb. 11 in the journal Cell Stem Cell, a team led by cardiologist Timothy J. Kamp reports transforming mouse fibroblasts, cells found mostly in connective tissue such as skin, into primitive master heart cells known as induced cardiac progenitor cells. The technology could permit a scalable method for making an almost unlimited supply of the three major types of cells in the heart. If replicated in human cells, the feat could one day fuel drug discovery, powerful new models for heart disease and the raw material for treating diseased hearts.
The lead author of the new study, UW-Madison postdoctoral fellow Pratik A. Lalit, found that 11 genes that play a central role in embryonic heart development could be used to reprogram the fibroblasts. He and his colleagues then narrowed the number of essential genes to five. Importantly, the group also defined the conditions necessary for the transformed cells to be effectively cultured in the laboratory.
Postdoctoral fellow Pratik Lalit, lead author of the new study.
Using the five genes, Lalit, Kamp and their team could push the fibroblast cells back in developmental time to become the cardiac progenitor cells that make cardiomyocytes, smooth muscle cells and endothelial cells — the trio of workhorse cells that make up the organ. The induced cardiac progenitor cells are capable of making billions of the critical heart cells, providing ample material to study heart disease in the laboratory dish, equip high-throughput screens to test various compounds for safety and efficacy, and ultimately, to treat heart disease by replacing diseased cells with healthy ones.
“Because the reprogrammed cells are actively dividing, we can generate billions of cells with relative ease,” says Kamp, who also co-directs the UW-Madison Stem Cell and Regenerative Medicine Center.
The study, explains Lalit, was like an exercise in reverse engineering: observing the genetic factors in play as the heart develops in a mouse embryo and using those to direct the fibroblast down the cardiac developmental pathway or lineage. “We’re learning from what happens in the embryo during cardiac development,” he says. “What does it take to make a normal heart?”
Induced cardiac progenitor cells (iCPCs) injected into hearts of mice with experimentally induced heart attacks generate new heart muscle. Newly developed heart muscle cells are shown by overlapping red (heart muscle protein) and green (iCPC protein) labeling, and cell nuclei are shown in blue. Image: Pratik Lalit
A key advantage of the engineered cardiac progenitor cells, notes Kamp, is that unlike all-purpose pluripotent stem cells, which can become any of the 220 different kinds of cells in the human body, the induced progenitor cells made from fibroblasts are faithful only to the cardiac lineage — a desired feature for cardiac applications. A potential drawback of cell transplants derived from all-purpose stem cells is the small but very real possibility of creating a teratoma, a tumor from tissue other than the intended cell lineage.
“With cardiac progenitor cells, you can reduce the risk of tumor formation as they are more committed to the heart lineages and are unlikely to form a tumor,” says Kamp.
If replicated in human cells, the feat could one day fuel drug discovery, powerful new models for heart disease and the raw material for treating diseased hearts.
Lalit and Kamp’s team tested the new cells in mice by experimentally inducing heart attacks. Injecting the engineered cells into the damaged hearts of mice, they observed the cells migrating to the damaged part of the heart and making cardiomyocytes — the heart cells that contract to underpin the beating of the heart — as well as smooth muscle and endothelial cells, key cells that form blood vessels. The implanted cells led to an uptick in survival of the heart-impaired mice.
The work was completed by a team of Wisconsin investigators, funded through the National Heart, Lung and Blood Institute’s Progenitor Cell Biology Consortium, part of the National Institutes of Health, and the American Heart Association. Contributing to the work were scientists from the University of Minnesota.
In October, when David O’Connor last visited Brazil as part of a decade-long research program studying drug-resistant strains of HIV, one of his Brazilian collaborators had a request.
“He asked about using some of the technologies we have developed to look for new viruses to study some unusual cases of a birth defect, microcephaly, in the north of Brazil,” says O’Connor, a University of Wisconsin–Madison pathology professor.
The babies born with underdeveloped brains and small heads were the relatively quiet beginning of worry over the spread of Zika virus, concern that has grown louder outside Brazil with an international outbreak and emergency attention from public health officials around the world.
“At the time we didn’t know it would explode into the public consciousness like it did,” O’Connor says. “But we did start planning.”
That planning will soon culminate in some of the first experiments studying Zika virus in monkeys, conducted by a broad UW–Madison team that includes the Wisconsin National Primate Research Center and expertise in infectious disease, pregnancy and neurology.
Pathobiological sciences Professor Jorge Osorio and research scientist Matthew Aliota, who were first to identify the Zika virus circulating in Colombia in October, provided essential Zika virology expertise. Ted Golos, professor of obstetrics and comparative biosciences, studies how other infections during pregnancy impact newborn health. The research group has extensive experience with viruses in humans and nonhuman primates — such as HIV and influenza — and their work will be conducted in secure facilities designed for the safe study of potentially harmful viruses.
Zika virus is transmitted by a specific mosquito called Aedes aegypti. Photo: James Gathany/Centers for Disease Control and Prevention
Their work will start with basic questions about Zika virus infection. Very little is known about the virus even though more than 50 years have passed since it was discovered in the Zika Forest in Uganda.
Until recently, Zika was expected to cause little more than flu-like symptoms — the Centers for Disease Control and Prevention lists fever, joint paint and a headache — in about 20 percent of the people it infected.
“That’s why it’s an understudied virus,” O’Connor says. “The viruses that get the most attention are the ones that makes us the most sick.”
The rapid spread of the virus and potential connection to an otherwise rare birth defect have drawn plenty of attention from the public and from government officials.
“People want clear answers, and we want to be able to make clear public health recommendations,” says Thomas Friedrich, a UW–Madison professor of pathobiological sciences. “There are a lot of countries in the tropics right now saying, ‘Don’t get pregnant until 2018.’ That’s not a sustainable public health recommendation.”
This is a digitally-colorized transmission electron micrograph (TEM) of Zika virus. Virus particles are colored red. Image: Cynthia Goldsmith/Centers for Disease Control and Prevention
In January, the National Institutes of Health made Zika virus research a high priority, and the groundwork underway at UW–Madison led to NIH support for a series of studies of the virus in macaques, monkeys whose physiology and immune systems are similar to humans.
The researchers will track the effects of initial infections, but also try to establish whether one Zika virus infection provides some protection against future infection — like chicken pox does.
Zika does not mutate particularly fast, the feature of HIV and influenza that makes those viruses hard to pin down with vaccines (like HIV) and leaves people open to reinfection seasonally (like flu). This may make Zika easier to head off with a vaccine, but the best sort of immune response to provoke with a vaccine is not yet known.
“That’s why we need to have data that shows what natural immunity looks like and the sort of immune responses that arise to protect an individual when they encounter that virus again,” O’Connor says.
Perhaps more hotly anticipated will be results from planned studies of Zika and pregnancy.
“We strongly suspect Zika infection during pregnancy is associated with birth defects such as microcephaly,” Friedrich says. “But we don’t know how strong the link is, or what percentage of women who get infected might give birth to children with birth defects.”
“The key messages are that we don’t know a lot. We will know a lot 12 months from now. But it’s really important we let data guide the decision making, not our guts.”
Or whether the timing of the infection during pregnancy matters. Or whether it is direct infection of a developing fetus by the virus or the immune response the infection sparks in pregnant women that causes problems like microcephaly.
“There are questions that cannot be safely and ethically addressed in humans that are absolutely vital,” O’Connor says. “What we will learn about Zika from the monkeys will hopefully have an immediate application when figuring out how to deal with this from a public health perspective.”
Working with the Zika virus from the original 1947 discovery in Africa and from the ongoing South American outbreak — provided by UW–Madison Osorio and Aliota, who were first to identify Zika virus circulating in Colombia in October — the researchers also hope to identify any important differences in infection by the different strains.
O’Connor also hopes results from the studies will help settle minds around the world, and help change the tenor of Zika news stories.
“The more hyperbolic the media coverage is, the more it gets repeated, reposted, retweeted,” he says. “The key messages are that we don’t know a lot. We will know a lot 12 months from now. But it’s really important we let data guide the decision making, not our guts.”
Each year, thousands of newborn babies suffer complications during pregnancy or birth that deprive their brains of oxygen and nutrient-rich blood and result in brain injury. This deprivation results in hypoxic ischemic encephalopathy (HIE), which can lead to long-term neurological issues such as learning disabilities, cerebral palsy or even death.
Researchers have known for some time that male infants are more vulnerable to HIE than females, but why this gender difference exists has remained a mystery.
In a study published this week in the journal eNeuro, researchers at the Waisman Center at the University of Wisconsin-Madison, led by Pelin Cengiz, associate professor in the Department of Pediatrics, show that a particular protein found in the brains of both male and female mice is present at higher levels in females, which offers them stronger protection against this type of brain injury.
“People often think that biological sex differences start to arise only after puberty, but they actually start in the womb and persist until the tomb,” says Cengiz, paraphrasing a 1999 statement by the Institute of Medicine. “So, treatment approaches that may work for newborn boys may not work for girls, and vice versa. We need to get it right to develop effective therapies.”
The protein is called estrogen receptor α, or ERα for short, and the researchers set out to learn how it confers its gender-specific protective effects.
Their first clue lay with a particular drug known to protect female but not male newborn mice from the effects of brain injury caused by HIE. The drug works by turning on a cascade of protective effects in the brain in response to oxygen deprivation and reduced blood flow.
Green shows the activation of a protective protein (TrkB) after hypoxia-ischemia in the brain (hippocampus) of a newborn female mouse. This green signal is not seen in the brain of female mice that are lack the ERα protein. Image courtesy of Pelin Cengiz
The team learned that, like the drug, ERα also causes a similar cascade in infant mice and the protein is actually required for the drug to be effective. The researchers found that female mice lacking the ERα protein could not activate protective factors following HIE, even when treated with the drug.
When the researchers studied the brains of male and female mice that could make the ERα protein, they learned that levels of this protective protein were significantly higher in female compared to male brains following oxygen deprivation and reduced blood flow.
“Under normal circumstances the brains of male and female mice have similar amounts of ERα,” says Cengiz, who is now exploring why ERα levels increase in female but not male brains after HIE.
Understanding the mechanism of how female brains are more resistant to damage from oxygen deprivation and reduced blood flow is a first step toward helping newborns of both sexes recover after suffering from HIE and live functional lives. It could also lead to more effective therapies and treatments for both genders, Cengiz says.
But more work needs to be done. For one thing, Cengiz and her colleagues looked at only the hippocampus region of the brain, which is linked to memory and learning and is involved in other neurological roles. The hippocampus is also a site where new neurons are continually generated throughout the lifespan.
“We focused on the hippocampus because we see memory and learning disabilities in many of the children affected by HIE,” says Cengiz, “and it is also the part of the brain that is most often injured after HIE.”
While it could be years before human babies benefit, each molecular mystery researchers unravel provides a potential new road to developing new therapies, Cengiz says, noting: “We are driven by the desire to improve outcomes for all newborns who suffer brain injury from HIE.”
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