A Mathematical Model for Marathon Running

Benjamin Rapoport had only six miles to go in the 2005 New York City Marathon when he felt his energy level drop dramatically — a sensation known to marathon runners as “hitting the wall.” “You feel like you’re not going anywhere,” Rapoport said of the experience, in a Massachusetts Institute of Technology (MIT) press release. “You can’t will yourself to run any faster.”

Rapoport was not alone. According to one recent study, more than 40 percent of marathon runners hit the wall, as Rapoport did. About 1 to 2 percent of race participants drop out before reaching the finish line, 26.2 miles from the starting line.

Now, however, thanks to Rapoport — who also happens to be an M.D.-Ph.D. student in the Harvard-MIT Division of Health Sciences and Technology — marathon runners can use a mathematical model to develop a personalized plan for avoiding the dreaded “wall.” Rapoport describes his mathematical model in the October 2010 issue of the journal PLoS Computational Biology.
Hitting the Wall

The experience of hitting the wall, or “bonking” as it is sometimes called, is not the result of simple fatigue or lack of motivation. Rather, it is a physiological response that occurs when the body’s carbohydrate supply runs out.

The human body uses both fats and carbohydrates as sources of energy. However, the process of metabolizing carbohydrates (that is, breaking them down for energy) is more efficient and requires less oxygen than metabolizing fat. Therefore, when the body has an immediate need for vast amounts of energy — during a marathon, for example — it relies heavily on carbohydrates. The main sources of carbohydrates available to a marathon runner include glycogen, a starch, stored in the leg muscles and the liver, and a small amount of glucose, a sugar, in the blood.

If the body’s carbohydrate stores run out during a marathon, fat becomes the body’s only available energy source. At this point, runners notice a significant drop in energy level: in short, they hit the wall. As they continue to run, their sense of physical exhaustion becomes even worse; fat metabolism produces by-products called ketones which accumulate in the body, resulting in increased pain and fatigue.
Modeling the Marathon

Some runners view hitting the wall as an inevitable part of running a marathon. Others try to avoid running out of carbohydrates by “carbohydrate loading” before the race — that is, eating a diet low in carbohydrates for a period of time, and then shifting to a high-carbohydrate diet in the days immediately before the race. However, as Jake Emmett, a professor of kinesiology and sports studies at Eastern Illinois University states in the MIT press release, “There is a lot of guesswork out there about carbo-loading and about carb intake during a marathon.”

Rapoport decided to take the guesswork out of the process by developing a mathematical model that determines how a runner can avoid hitting the wall. His model involves two key factors that influence metabolism: exercise intensity and the amount of glycogen that can be stored in the runner’s leg muscles.

Exercise intensity is essentially a measure of how hard the body is working while performing a certain exercise. It can be defined as the ratio of the athlete’s heart rate — measured while the athlete is engaging in a particular exercise — to the athlete’s maximum heart rate. Exercise intensity is a key factor in Rapoport’s model because it determines how much of the body’s energy is being drawn from carbohydrates versus how much is being drawn from fats. As Rapoport notes in his paper, “carbohydrates [account for] a greater proportion [of energy] at high intensities.”

If runners want to avoid hitting the wall during the marathon, they must run at a slow enough pace — maintaining a low-enough exercise intensity — to allow the body to achieve the right balance between fat and carbohydrate metabolism. Specifically, the proportion of fats to carbohydrates metabolized should be fine-tuned so that the body’s carbohydrate stores last until the very end of the race. Rapoport’s model allows runners to pinpoint the exact pace they should maintain to achieve this goal.

The second key component in Rapoport’s model is the leg muscles’ ability to store glycogen. Glycogen in the leg muscles constitutes a large portion of the carbohydrates metabolized during a marathon. However, the amount of glycogen in these muscles can vary significantly from one runner to the next, depending on leg muscle size and density. Using Rapoport’s model, runners can determine how their muscles’ capacity for storing glycogen can influence their ideal pace and the amount of carbohydrates they should consume, either before or during the race.
Trying It Out

Rapoport has tested his model using data for typical male and female marathon runners, and his results provide strong evidence that the model is accurate. According to the model, an 18-to-34-year-old man who has been training intensively for a marathon can expect to finish the race in 3 hours and 10 minutes without hitting the wall. This turns out to be the exact time that male runners in this age group must achieve to be eligible to participate in the Boston marathon. Similarly, Rapoport’s estimated time for a female marathon participant in this age group exactly matches the Boston Marathon’s qualifying time of 3 hours and 40 minutes.

Marathon runners interested in finding out their ideal pace, along with personalized guidelines for carbohydrate consumption before and during the race, can consult Rapoport’s paper. Rapoport’s model is also available in the form of a simple online calculator at endurancecalculator.com/ index.html. The calculator requires the user to enter a few basic pieces of information such as age, heart rate (which can be used to estimate exercise intensity) and weight (which can be used to estimate leg-muscle size and density).

For marathon runners, Rapoport’s model is a long-awaited tool that answers important questions about how to pace oneself during a marathon. For those of us who have never participated in a marathon, we can only hope that Rapoport’s next paper will answer yet another pressing question — how to run 26.2 miles without all of the hard work.

Discussion Questions

Do you think the calculator described here would work equally well for other endurance sports, such as long-distance cycling or swimming? Why or why not? The calculator uses age, heart rate, and weight as data; are there other types of health/exercise related data that you think would be relevant? How might you try to assess — in a controlled and ideally quantitative way — the relative importance of the factors used in calculating an ideal running pace?

How about running insoles? Can stopping shock and giving your feet more bounce really help with running? Many specialist recommend wearing special insoles (for example), however an equal amount of other specialists discourage their usage.

New Hope for Blast-Damaged Brains

When media outlets report on wartime injuries, footage of soldiers with missing limbs often makes the nightly news and stories on post-traumatic stress disorder grab headlines. However, the most common wounds suffered by military personnel serving in Iraq and Afghanistan have been brain injuries. Traumatic brain injuries (TBI) occur when soldiers are exposed to blasts from improvised explosive devices and roadside bombs. Some sources estimate that almost 16% of all soldiers in both war zones have experienced a mild brain injury from an explosive blast. These injuries are often different from brain injuries suffered by civilians, and the medical community has few effective treatments for them.

After serving a tour of duty in Afghanistan, Kevin Kit Parker, a major in the U.S. Army and a bioengineer at the Harvard School of Engineering and Applied Science in Cambridge, Massachusetts, was inspired to conduct research into brain injuries caused by bomb blasts. In two recently published papers, his lab identified the biological mechanisms that cause neurons to swell and break, and blood vessels to shrink, when brain tissue is damaged in an explosion. The damage is caused by a cell signaling pathway, called the ROCK pathway, which runs amok after the blast. The researchers hope that post-blast treatment with a drug that blocks the pathway from forming may prevent some of the damage. The findings were published in the Proceedings of the National Academy of Sciences, and in PLoS One.

“So many young men and women are returning from military service with brain injuries, and we just don’t know how to help them,” said Parker in a press release. “We have established a toe-hold as we try to climb up on top of this problem. In many ways, this work is just the beginning.”
Blast Research Comes to a Head

A traumatic brain injury (TBI) may occur off the battlefield — for example, in sporting events and car accidents, and in shaken baby syndrome. The injury occurs any time there is an impact or force that shakes the brain or penetrates the skull and disrupts the brain’s normal function. TBIs may range from mild concussions to loss of consciousness followed by amnesia.

TBIs may have a variety of immediate and long-term effects: they may impair the ability to think clearly, as well as the senses of taste, touch and smell. Victims’ communication and language skills, and their emotional state, may also be affected. TBI is also known to cause epilepsy in some cases, and has been linked to Alzheimer’s, Parkinson’s and other degenerative brain diseases.

The type of TBI that has been occurring in soldiers in the wars in Iraq and Afghanistan is called blast-induced TBI; it is caused by the detonation of improvised explosive devices and roadside bombs. These blast forces may cause only mild TBI, but they can still have long-term effects on military personnel. The symptoms often differ from those commonly experienced by civilians, and currently there are few medical treatments specifically designed for blast-induced TBI.

Suffocating Cells

To improve medical treatment for TBI in military personnel, Parker and his lab developed a system that simulates the effects of TBI on nerve cells and blood vessels in the brain. They cultured layers of neurons or blood vessel tissue and attached them to a stretchy polymer. Then, using a “high velocity tissue stretcher” that they had built, they quickly pulled on both sides of the stretchy material to simulate a blast wave going through the cells.

In an article published in the Proceedings of the National Academy of Sciences, the researchers reported that vascular smooth muscle cells — the type of cells that line blood vessels — could account for one common side effect of blast-induced TBI, called cerebral vasospasm. This is a condition where the blood vessels surrounding the nerve cells contract, cutting off the blood supply to the cells. Vasospasm exacerbates brain damage by starving neurons of oxygen. It had long been assumed that vasospasm occurred only when there was hemorrhaging inside the brain, but, since the start of the war in Afghanistan, vasospasm has often been observed in blast-induced TBI with no bleeding.

After giving the smooth muscle cells a carefully measured tug, the researchers studied them for 24 hours to look for changes. The cells appeared intact under the microscope, but an hour after the blast they began taking up high concentrations of calcium ions from the surrounding environment. The calcium ions interacted with molecules called actin, which make up the cytoskeleton inside the cell. In all eukaryotic cells, actin molecules assemble into filaments and form a scaffolding, which determines the cell’s shape and allows it to move. When actin was stimulated by the increased calcium concentration in the muscle cells, it caused the entire cell to contract, resulting in vasospasm.

Parker attempted to prevent vasospasm in the cells by treating them with a drug that blocks the signaling pathway that is known to regulate cell contraction. A regulator called ROCK controls the movement of actin filaments. The researchers found that when they treated a layer of blasted cells with a drug that blocks ROCK, called HA-1077, vasospasm could be prevented.

The activation of the ROCK pathway and its effect on cell contraction explains why military doctors often observed vasospasm in mild cases of TBI even when they found no evidence of hemorrhaging. “They’ve duplicated [in cell culture] a finding that has been baffling to clinicians,” is what Jack Tsao, a neuroscientist at the Uniformed Services University of the Health Sciences in Bethesda, Maryland, told ScienceNOW.
When the Network Breaks Down

In a second paper, published in the journal PLoS One, the researchers used rat neurons to simulate the damage seen in the nerve cells of soldiers with blast-induced TBI. After “blasting” the rat neurons with their tissue stretcher, they found that the axons — long projections from the neuron that attach to other cells and transmit signals — were broken in some places and had large swellings in others.

The swellings occurred at places along the axons where the cell attached to other cells, or the extracellular matrix (ECM), which is the connective tissue that supports the neurons. At these focal points, molecules called integrins anchor the axon by connecting the actin cytoskeleton inside the cell to the ECM or to the membrane of another neuron. When the integrins are dislodged by a blast, molecular signaling inside the cell runs amok and causes the actin filaments to contract. The contraction pulls in the axons, causing swelling, and breaks up the neural networks that make up the brain.

Since integrin is also regulated by ROCK, the researchers treated the blasted neurons with the same drug. “Encouragingly, we also found that treating the neural tissue with HA-1077, which is a ROCK inhibitor, within the first 10 minutes of injury, reduced the number of focal swellings. We think that further study of ROCK inhibition could lead to viable treatments within the near future,” said Borna Dabiri, a bioengineering graduate student in Parker’s lab, and the lead author on the PLoS One paper, in a press release.

“This is pathological activation of a totally healthy signaling pathway,” said Parker to Nature News. Fortunately, since the same pathway causes the blood vessels and the neurons to contract, the same drug can be used to treat both problems, says Parker. “That’s a really a rich therapeutic opportunity.”
New Treatments for Battlefield Blasts?

Parker cautions that these findings are very preliminary, and notes that further studies will be needed to see if the cells in a soldier’s brain react like the cells in his culture plates. “It would be inappropriate to extrapolate from a dish to some dude’s head,” Parker commented to ScienceNOW. He also hopes to try smaller forces, such as those that would cause a mild concussion, and wants to explore different cellular pathways before conducting drug testing in animals and humans.

Ken Barbee, a bioengineer who studies neuron injury and repair at Drexel University in Philadelphia, Pennsylvania, is intrigued by the paper, but remains unconvinced that the ROCK pathway is the cause of the cellular changes. In his own work, he found that tears in the cell membrane of neurons can cause TBI, and has successfully used drugs to repair the tears. “I think the reality is that when the whole tissue undergoes deformation, you’re probably going to get a combination” of injuries to the neurons and disrupted cell signaling pathways, Barbee commented to Nature News. When asked about Barbee’s claim, Dabiri agreed that membrane tears are common in cases of severe TBI, but claims that they have not been observed in mild TBI cases, such as those simulated by their experiments.

The group’s findings have been hailed by a number of other scientists studying TBI, and their approach is seen as providing new techniques for TBI research. “It’s an elegant demonstration that biomechanical stretch will produce changes in the cytoskeleton,” is what David Hovda, director of the Brain Injury Research Center at the University of California, Los Angeles, told Nature News. “This is an important fundamental discovery.”
Discussion Questions

Would you expect soldiers who have experienced blast-induced TBI to be likely to also suffer from post-traumatic stress disorder? How does the chaos on the battlefield complicate blast-induced TBI treatment? Do you think HA-1077 would be effective in treating TBI caused by sports mishaps or car accidents?

Soccer Heading: A Memory Issue

Soccer players beware: you might want to think twice before heading the ball.

A study published in the June 11, 2013 issue of the journal Radiology, finds that players who head the ball frequently score worse on memory tests and are at higher risk of brain abnormalities than those who head the ball less often.

In soccer, “heading” is an offensive or defensive move in which a player uses his or her unprotected head to deliberately strike the ball and direct it during a game. On average, most players head the ball 6 to 12 times during competitive games, where balls can travel up to 50 miles per hour (80 kilometers per hour). Heading is even more frequent during practice sessions: it’s not uncommon during heading drills for players to perform 30 or more headers in a short span of time.

The effects of sports-related concussions on cognitive functions have been well documented, and substantial media attention has been devoted to the cognitive impairment seen in retired football players in particular. But the clinical significance of less severe “subconcussive” impacts such as those associated with heading in soccer has not been investigated as thoroughly, and there is no consensus on how dangerous such impacts may be.
A Heading Threshold

To investigate the effects of subconcussive trauma on brain function, neuroradiologist Michael Lipton and his colleagues performed diffusion tensor imaging (DTI), an advanced MRI-based imaging technique that measures the uniformity of water movement (called fractional anisotropy) throughout the brain on 28 men and nine women soccer players (median age 31) recruited from amateur leagues in New York City. They also gave the players memory tests.

Lipton, who is at the Albert Einstein College of Medicine in New York City, and his team hypothesized that they would detect a “threshold” for heading, above which the risks of brain abnormalities and memory impairments would increase. They believed that if their hypothesis were confirmed, it would have major consequences for public health, the authors write in their paper.

The soccer players in the study were asked to recall about how many times they had headed the ball over the past 12 months. Most of them reported having done so hundreds of times, and the count for some players was in the thousands. The median heading count for the group was 432, while the range was from 52 to 5,400.
Brain Abnormalities

When the researchers looked at the DTI scans of the players, they found that some of them showed brain abnormalities. The abnormalities occurred mainly in the white matter of three regions of the brain that are associated with attention, memory, sensory inputs, visual and spatial processing, and other cognitive functions.

The team also found evidence of thresholds for damage in the three brain regions investigated, thus confirming their hypothesis. One brain region, for example, had a header threshold of 850 — that is, players who headed the ball at least 850 times were more likely to show abnormalities in this part of the brain than those who fell short of the threshold. For the other two brain regions, the thresholds were about 1,300 and 1,550 headers.

The affected brain regions lie near the back of the head—opposite the typical point of impact of a header. This might seem counterintuitive at first, but the researchers hypothesize that brain “recoil” could explain the finding “When there is a head impact, the brain sloshes back and forth inside the head,” Lipton told ScienceNews. When a player performs a header with the front of the head, Lipton added, the brain presses against the front of the skull momentarily but then slams into the back of the skull.
Memory Impairments

The researchers also conducted memory tests on the players. They found that the nine players with the highest header count scored worse on average than the nine who had the fewest. The researchers estimated that the threshold for memory loss was about 1,800 headers.

The new findings add to a growing body of evidence that not only sports-related concussions but also less severe “sub-concussions” can lead to long-term memory impairment, reduced attention span and impaired concentration. “These changes are subtle,” Inga Koerte, a radiologist at Harvard Medical School and Brigham and Women’s Hospital, both in Boston, Massachusetts, told ScienceNews. “But you don’t need a concussive trauma to get changes in the microstructure of your brain,” said Koerte, who was not involved in the study.

In a previous study, Koerte had found that even soccer players who had never been diagnosed with a concussion had more white matter abnormalities than swimmers did. “While we do not yet know what these changes mean, soccer players should be aware that they may be putting themselves at some risk for developing brain injuries,” Koerte said in a recent interview with the German Center for Research and Innovation.

The discovery of header thresholds for brain abnormalities and memory impairment could be used to create safety guidelines for soccer players, Lipton and his coauthors write in their paper. For example, “prospective monitoring of exposure at the team level, perhaps to be termed head counts, could identify a point at which a player’s heading should be curtailed for a specific recovery period,” they write. The researchers noted that such an approach is already being used in youth baseball, where pitchers are limited to a certain number of pitches to reduce the risks of arm and shoulder injuries.

Lipton and his colleagues also envision genetic tests that players could take to help reduce their risk of brain injuries. For example, a player found to have a genetic variant that had been linked to a greater risk for heading-related traumatic brain injuries might be advised not to head. The authors observed that further research would help in developing “evidence-based protective strategies that can ensure the future of safe soccer play.”
Discussion Questions

The soccer study asked players to report on how much heading they had done. Many studies involve such “self-reporting,” but questions can be raised about self-reported data. What problems might be associated with self-reporting? How might one try to obtain heading data without relying on self-reporting?

The study correlates possible negative consequences of heading with the number of times the ball had been headed within a certain period of time (in this case a year.) How might one test whether the overall number of headers is the most important factor, as opposed to, say, differences in players’ skulls or other physical features. Heading the ball has also been linked to neck injuries which you can read about on thesciencepost.

Human Stem Cells Made by Cloning

Wouldn’t it be great if every time a patient needed a new kidney, a stronger heart or a batch of new brain cells scientists could simply grow the required spare part in a petri dish? While a few decades ago this idea would have sounded like a plot for a science fiction novel, it may very well become standard procedure within decades thanks to the discovery of stem cells.
There are about 300 different types of regular “somatic” cells in the body. (Somatic cells make up most of the body’s tissues—the term is used in contrast to cells, such as sperm and egg cells, that serve reproductive purposes.) Each type of somatic cell has unique properties that allow it to perform a particular function. For example, muscle cells contract and expand to allow us to move, while nerve cells transmit signals that let us experience the world around us, recognize familiar faces, memorize poems, and so forth.

While somatic cells are not interchangeable—you wouldn’t expect a photoreceptor cell from an eye to drive muscle contraction or a skin cell to transport oxygen—stem cells lack a specific identity and can develop into different types of tissue. The so-called embryonic stem cells make up the blastocyst, which forms when an egg cell is fertilized and eventually gives rise to all of the different tissues of the body. Like a blank tile in Scrabble that can turn into any letter you want, an embryonic stem cell has the potential to become any type of body cell. However, once it does, there’s no turning back: just like the wildcard Scrabble piece that can only be used once, a stem cell is locked into its “chosen” identity.

While embryonic stem cells are present only in the earliest stages of development, certain non-embryonic varieties stay with us through adulthood. Non-embryonic stem cells are more specialized and allow the body to repair itself. For example, bone marrow, blood, and even the brain have some natural capacity for regeneration.

The unique flexibility of stem cells makes them a godsend for regenerative medicine. Like a customized tissue factory, they can be used to produce a constant supply of replacement cells genetically identical to the recipient. However, even though scientists have been looking for ways to put stem cells to medical use for over a decade, the task has been difficult due to technical complications, legal issues and ethical dilemmas. In a recent issue of Cell, scientists from the Oregon Health and Science University (OHSU) describe how they were able to create human embryonic stem cells via cloning. Their results are a milestone in what scientist John Gearhart calls “a holy grail that we’ve been after for years.”
Crime or Cure

For the last few decades, doctors have been able to use certain types of stem cells to treat certain diseases. For example, since bone marrow contains stem cell precursors to different cells found in blood, bone marrow transplants can help treat a number of different blood-related disorders, such as leukemia. While treatments using embryonic stem cells are still experimental, one such approach recently restored sight to a man whose vision went from 20/400 to 20/40. Another team of scientists managed to use antibodies to “reprogram” bone marrow stem cells to give rise to brain cells.

And this is just the beginning: the potential benefits of using stem cells in other medical procedures are almost inexhaustible. Doctors may someday be able to use them to treat anything from Alzheimer’s or Parkinson’s disease to heart disease, cancer, or even baldness. It’s hard to think of a medical condition that wouldn’t offer some point of attack for this potentially revolutionary tool.

While the field of regenerative medicine is very promising, it is also one of the most controversial areas of research, marked by technical complications, ethical dilemmas, and even scientific fraud. The main sticking point is the use of human embryos to harvest stem cells—a task first accomplished by a University of Wisconsin team in 1998—and the fact that the embryos are destroyed in the process. Even though the embryos used in research are usually ones discarded from in vitro fertilization, some people feel, on religious and ethical grounds, that this is wrong, and consequently the research became a political football, with some arguing that embryonic stem cell research should be federally funded since it held such great promise, and others contending that federal funding of such research would be a grave mistake.

To bypass some of the ethical dilemmas, scientists began to look for alternative ways of harvesting stem cells. A major advance came a few years ago, when Gladstone Institute of Cardiovascular Disease scientist Shinya Yamanaka developed the first “induced pluripotent stem cells” (referred to as iPS). Unlike embryonic stem cells, iPS cells are derived from regular somatic cells that get “reprogrammed” back to their undifferentiated state.

However, while Yamanaka’s discovery earned him a Nobel Prize in 2012 and provided an option acceptable to both sides in the stem cell debate, it remained unclear whether or not iPS cells are really just as good as embryonic stem cells. Critics point out that iPS cells are created using retroviruses, which may cause potentially dangerous genetic abnormalities. Moreover, iPS cells tend to age quickly and don’t last very long.
Hello Again, Dolly

While some researchers continue trying to improve iPS, others have turned to a third method known as somatic cell nuclear transfer (SCNT). The technique, also referred to as “therapeutic cloning,” allows scientists to create embryos by swapping the genetic material of an egg cell for the DNA of another person—for example, that of a sick patient.

In nature, an embryo develops when an egg cell is fertilized by a sperm cell, both of which are “haploid” and have half the total number of chromosomes found in somatic cells. However, by replacing the modified egg cell’s genes with a full set of chromosomes from a body cell a researcher can “trick” the egg into acting as if it had been fertilized. As a result, it starts dividing and grows into an “embryo” genetically identical to the body cell’s donor.

The overall idea isn’t a new one: 17 years ago, Roslin Institute scientist Ian Wilmut used this method to create Dolly, the famous cloned sheep. Since then, enough species have been cloned to fill a whole zoo— for example, Dewey the deer at Texas A&M University; Snuppy, a South Korean Afghan Hound; two ferrets, Libby and Lilly; Ralph the rat; a mouse named Cumulina—among many others.[See Adult Mammal Cloned for First Time, April 1997; On the Heels of Dolly Come Polly and Molly, February 1998; 5 Cloned Piggies, All in a Row, May 2000; Doggy Double: Korean Scientists Create First Canine Clone, August 2005.]

However, the process didn’t seem to work for human egg cells, which are more fragile than those of other species. The search for ways around this obstacle turned out to be very messy. When Seoul National University veterinary scientist Hwang Woo Suk published reports of successful SCNT trials with human cells in 2004 and 2005, it seemed that finally a solution had been found. However, what looked like a groundbreaking discovery turned out to be one of the most infamous cases of scientific fraud, with falsified data underlying the claims. [See Hwang Woo Suk and Fraud in Science, January 2006; Hwang Woo Suk: Seeing Double, December 2005; Scientists Clone Human Embryos to Create Stem Cells, February 2004.]

While other efforts to produce human stem cells by using the SCNT method have been more legitimate, they failed to hit the mark due to technical complications. For example, New York Stem Cell Foundation specialist Dieter Egli produced a line of human stem cells in 2011. However, since the egg’s nucleus remained in the cell, the resulting stem cells had an abnormal number of chromosomes.
Breakfast of Champions

Shoukhrat Mitalipov, senior scientist at the Oregon National Primate Research Center and lead author of the Cell paper, has been investigating stem cells since the 1990s. In 2007, his team used the SCNT method to clone embryonic stem cell lines in monkeys. The scientists then used knowledge gleaned from those studies to successfully clone human embryonic stem cells, the feat described in the Cell paper.

Mitalipov took egg cells that had been donated by volunteers to serve as the basis for future stem cells. Since egg quality is essential for this process to work, the eggs came from healthy, young donors. He then cleared out the DNA from the cells and replaced it with DNA from the skin cells of other individuals (the first DNA came from fetuses and the rest came from an eight-month-old with Leigh syndrome, a rare metabolic disorder). “The idea is that the egg cytoplasm has…the ability to reset the cell’s identity,” Mitalipov explained in an interview with Fox News. “It basically erases all this memory, and now we can derive them and make them into stem cells.”

From previous experience, Mitalipov knew he had to be extra careful with the DNA exchange process, timing it to take place when the egg was most likely to accept the new genetic material. An inactivated Sendai virus helped fuse the egg with the body cells, and an electric jolt helped jump-start embryonic development.

An ingredient that played an important role was, oddly enough, a substance we ourselves often rely on to get going in the morning—caffeine. Since caffeine inhibits an enzyme responsible for breaking down the “maturation promoting factor,” adding it to the mix helped prevent premature activation during spindle removal and somatic cell fusion. Harvard University scientist George Daley jokingly calls this “the Starbuck’s experiment.” “This little change in the cocktail,” he adds, “was what really allowed the experiment to really ultimately succeed.”

While each step was important, the most challenging part of the process was actually the combination of steps. As Mitalipov explains, “there is no one trick to making this work. It is like winning the lottery, all the numbers have to line up the right way to win.” Mitalipov’s monkey-cell research was especially useful for this purpose: before switching to human cells, which are more expensive and more difficult to obtain than monkey cells, Mitalipov fine-tuned the process for monkey cells by testing over a thousand combinations of steps.
Speedy Success

Having set up the cell colonies in December 2012, the scientists were excited to see that four of them started growing. The growth continued past the eight-cell stage, which had been the farthest scientists could go in previous attempts. Once the newly cloned embryos were five or six days old, Mitalipov and his team could manipulate them to create particular types of somatic cells.

Knowing how hard it has been for others to make this process work, Mitalipov was surprised to get results relatively quickly: “I thought we would need about 500 to 1,000 eggs to optimize the process and anticipated it would be a long study that would take several years. But in the first experiment we got a blastocyst and within a couple of months we already had an [embryonic] stem cell line.”

Mitalipov describes the successful outcome: “A thorough examination of the stem cells derived through this technique demonstrated their ability to convert just like normal embryonic stem cells, into several different types, including nerve cells, liver cells, and heart cells.” And while the technical details remain to be worked out, the team sees the results as “a significant step forward in developing the cells that could be used in regenerative medicine.”
Not So Fast

“Our finding offers new ways of generating stem cells for patients with dysfunctional or damaged tissues and organs,” Mitalipov explained in an interview. “Such stem cells can regenerate and replace those damaged cells and tissues and alleviate diseases that affect millions of people.”

The study was immediately hailed as an important breakthrough. However, a week later things took an unexpected turn. An anonymous commenter on PubPeer (a website that allows scientists to comment on the work of their colleagues) pointed out four mistakes involving images that had been mislabeled or duplicated.

Arnold Kriegstein, a researcher from the University of California, San Francisco, says that this situation “is really like déjà vu all over again.” But while it does add to the existing controversy surrounding the whole subject of stem cell research, the mistakes in Mitalipov’s paper are clearly accidental and don’t affect the validity of his results. More than anything, they appear to be the result of a rush to publish: the study was accepted by Cell within days of its submission.

In response to the criticism, Cell editors, as well as OHSU representatives, have called the inaccuracies “minor errors.” According to OHSU spokesman Jim Newman, “Neither OHSU nor Cell editors believe these errors impact the scientific findings of the paper in any way. We also do not believe there was any wrongdoing.”
Batch of Cells or Future Human?

But even if these details are ironed out, broader disagreements about Mitalipov’s approach are likely to persist. While raising hopes, the study has also stirred up ethical concerns that have been part of the stem cell debate from the beginning. As Daley puts it, “This is a huge scientific advance…But it’s going to, I think, raise the specter of controversy again.”

And, sure enough, it did. For one thing, using human embryos—even ones generated without fertilization—for medical research remains a bone of contention. When seen as a potentially revolutionary medical treatment, the procedure becomes more palatable. As University of Pennsylvania scientist John Gearhart says, “Where you can improve [a patient’s] quality of life tremendously through this kind of technology, I personally believe that it is ethical to use material like this.”

Moreover, Mitalipov argues that his method of obtaining stem cells is qualitatively different from one that uses fertilized embryos. However, he also recognizes that not everyone might agree with him: “We think it’s more ethically acceptable, but you never know. Some people [might] say now they’re trying to use cloned embryos.”

Indeed, those who believe that cloned embryos amount to potential human beings are hesitant to accept their use in the lab. The idea of “manufacturing” human life at any stage doesn’t sit well with everyone. Instead, opponents urge scientists to focus on methods that don’t involve embryos at all and argue that the discovery of iPS cells as an alternative way to produce stem cells should make methods involving embryos obsolete. Miodrag Stojkovic, a Serbian scientist and a fertility clinic director, is very frank in his assessment: “Honestly, the most surprising thing [about this paper] is that somebody is still doing human [SCNT] in the era of iPS cells.”

However, creating stem cells via cloning has significant advantages when compared with iPS. Instead of iPS’s reprogramming of the body cell of an adult, the nuclear transfer method provides a clean slate for the stem cells it creates. As Mitalipov explains, “If you take a cell from a 90-year-old patient, the battery is kind of drained. You could make new cells by reprogramming it, but the energy is gone.” However, “the idea behind using egg cells is that you’re fully recharging the battery and making cells that could probably live another 90 years. We’re taking you back to having some young cells.”

In order to get a more definitive answer about the way the two techniques compare, more studies comparing iPS and SCNT techniques have to be done. Along with team member Masahito Tachibana, Mitalipov plans to conduct a comparison study between the two methods using cells from the same donor.
The Clones Are Not Coming

While creating and destroying embryos at will might seem questionable to critics, allowing them to fully develop is even more unsettling. Cloning a batch of particular cells is a huge leap forward for medical science. However, cloning entire humans by using the same method could become a disaster.

In an effort to reassure people that this is not what he is doing, Mitalipov emphasizes the fact that his goals are solely “therapeutic,” rather than “reproductive,” cloning. The embryos he created will not be implanted: “We never tried that in humans and it’s not our intention,” he explains. Likewise, Daley insists that “no legitimate scientists would want to use this technology for reproductive purposes. They would see it not only as unethical, but unsafe and probably illegal.” Moreover, since, in Mitalipov’s work with monkeys, implanting cloned embryos didn’t produce viable offspring, Mitalipov argues that it’s very unlikely that this technique could be used for this purpose in humans.

However, just because Mitalipov isn’t planning to clone humans doesn’t mean nobody else is planning to try. It’s theoretically possible that someone will figure out how to use this method to clone humans in the future. (After all, SCNT itself didn’t initially seem to work for human cells.) “This study shows that human cloning can be done….The more important debate is whether it should be done,” said Richard Doeflinger at a meeting of the U.S. Conference of Catholic Bishops. Those attending the meeting were concerned that the findings “will be taken up by those who want to produce cloned children as ‘copies’ of other people.”

Stem cell research has been dogged by controversy from the start, and is likely to remain controversial for quite some time. If Mitalipov’s research leads to new therapies that are as significant as supporters of the research anticipate, that will certainly have a big effect on the debate.
Discussion Questions

Can you think of any explanations for why human cloning is more difficult than cloning other mammals?

Some cloned animals have had various problems, including shorter lifespans, as compared with animals born through natural processes—why do you think this is?

A factsheet on cloning from the National Human Genome Research Institute notes that telomeres (the tips of chromosomes) may have something to do with the shorter lifespans—why do you think this is?
Journal Abstracts and Articles

(Researchers’ own descriptions of their work, summary or full-text, on scientific journal websites).

“Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer” http://www.cell.com/abstract/S0092-8674(13)00571-0.

Cut-Off Point: Why Only Tips of Fingers Regrow

Lose a finger, and you’d better hope you can get it on ice. Lose a fingertip, and it might not matter. Fingertips are one of the few parts of the human body that can match the regenerative abilities of a starfish or a salamander. Children and some adults can grow new fingertips in a few weeks after amputation. The replacement fingertips wouldn’t pass for the originals; they lack fingerprints and their nails have flattened, square ends. Still, they have the full complement of bone, nerves and nails seen in regular fingertips.

This regeneration will occur only if enough of the fingertip is left intact. If any of the nail remains, even a sliver, the tip can regrow. Cut off the nail entirely, and neither it nor the fingertip will come back. Makoto Takeo of the New York University School of Medicine and colleagues investigated why nails were so crucial for regeneration, publishing their results online in Nature in June 2013. The key wasn’t the hard part of the nail, the nail plate, but instead the cells that produce it, the nail matrix. Looking at the tips of mice’s toes, the researchers found that the nail matrix gave off chemical signals that induced the regrowth of nerves and bone. Mice lacking these signaling molecules were unable to regenerate. Only the nail matrix cells closer to the tip, the ones below the nail plate, released the chemicals that enabled regeneration.
Nail Matrix

Ever wondered what that whitish crescent on your thumbnail is? It’s the nail matrix, or at least the part we can see. The rest extends underneath the skin at the base of the nail. If the matrix as a whole were what enabled regeneration, then it shouldn’t matter for amputees whether they have any nail left. An injury that removed the entire nail could still preserve some of the nail matrix, and the fingertip could still regenerate. Since this didn’t occur, it suggested that only certain cells in the nail matrix were involved in regeneration.

One possibility was that regeneration was governed by the nail stem cells. These slowly dividing cells produce the rest of the nail matrix, which goes on to form the nail plate and the nail bed. No one was sure where in the matrix the stem cells were located. To find out, the researchers examined genetically engineered mice known as reporter mice. These mice were designed to express reporter genes, genes that produce identifying chemical markers in particular cells. When a substance called tamoxifen was administered, cells making a protein involved in nail production expressed the gene for an enzyme called LacZ.

The researchers injected the mice with the synthetic chemical tamoxifen and examined their toes over the following months. There were streaks of LacZ activity underneath and behind the nail, but these faded as the cells divided. The distal portion of these streaks, the part closer to the tip of the toe, faded more quickly than the proximal portion toward the base of the toe. This indicated that the proximal cells were the slowly dividing nail stem cells. The distal ones were the more rapidly dividing cells that create the rest of the nail.
No Bones without Wnt

These distal cells appeared to be the best candidates for affecting regeneration. Was there anything in their biochemistry to support this? The researchers analyzed the patterns of gene expression in cells from different parts of the nail matrix. They found that the distal cells expressed genes involved in a set of chemical interactions called the Wnt signaling pathway. In embryos, Wnt signaling coordinates many aspects of development, including the formation of limbs and nails.

In the nail matrix, Wnt probably caused the distal cells to differentiate, changing them into the different types of cells in the nail. To verify this, the researchers prevented expression of β-catenin, a protein involved in the Wnt pathway. Since mice couldn’t develop normally without this vital protein, the researchers used another genetically engineered strain of mice called a conditional knockout. Just as the reporter mice produced an enzyme only under specific conditions, these knockout mice were genetically engineered to stop producing β-catenin in nail cells when the same chemical, tamoxifen, was administered. Two months after tamoxifen was given to these mice, they had stopped creating nails. In their place, the mice had a layer of cells resembling undifferentiated nail stem cells.

If Wnt signaling caused nail stem cells to differentiate into new nails, maybe it also caused tissue to differentiate into a new fingertip. The researchers took mice from the same strain of conditional knockouts and amputated the tips of their toes, so that only a small amount of nail remained. Unlike humans, normal adult mice will always regrow the tips of their digits. Some of the mice were treated with tamoxifen after amputation, stopping β-catenin expression and the Wnt signaling pathway. Others didn’t receive the tamoxifen injection. These control mice regenerated completely, growing new nails and toe bones in five weeks. The ones that had been given tamoxifen didn’t regenerate their nails or their bones.
Guiding Nerves, Growing Bone

How did Wnt signaling enable the mice to regenerate? A clue came from their nerves. As the control mice healed from their amputations, the nerves in their toes extended toward the tip of the regrown digit. The nerves made contact with the mesenchyme, the cells from which new bone forms. The tamoxifen-treated mice didn’t show this kind of nerve growth. New cells formed over the wound, but the mice’s nerves didn’t grow past the initial site of injury. The nerves never reached the mesenchyme, which consequently never differentiated into bone.
Mouse Toes

It’s well known that nerves are important for the regeneration of both rodent digits and salamander limbs. The researchers investigated what effect a lack of nerves in the tip of a digit would have on regeneration. In another group of mice, they severed the nerves leading to one foot and then amputated the tips of toes on that foot and the opposite one. The amputated toes on the feet with intact nerves would act as a control. As in the conditional knockout mice, the toes on the feet with severed nerves didn’t regenerate. The team found that these toes had low levels of FGF2 (fibroblast growth factor 2), one of a set of proteins that aid in wound healing and are essential for limb regeneration in amphibians. The conditional knockout mice with the amputated tips had lacked FGF2 as well. The researchers found other evidence for the importance of FGF2 when they grew cultures of mouse mesenchymal cells. When they administered FGF2, the cells divided more rapidly and began to turn into bone.
Restoring Wnt and Regeneration

If nail matrix cells could induce regeneration, then the researchers might be able to induce it artificially. The team repeated the severed nerve experiment. This time, after the tips of the mice’s toes were amputated, the researchers inserted beads soaked in FGF2 into the wounds. As FGF2 leached off of the beads, it spread into the mice’s tissues. After three weeks, they had begun to regrow bone.

Was it possible to give mice an improved ability to regenerate? The researchers amputated the tips of mice’s toes so that only the proximal part of the nail matrix remained. This meant that the injury was just past the point where the tip can normally grow back. The mice were genetically engineered so that, when treated with tamoxifen, their remaining nail stem cells produced β-catenin and initiated Wnt signaling. After the amputation and the tamoxifen injection, these mice made a full recovery, regrowing their nails along with new bone.

Regeneration is a complex process, but the researchers had identified two of its major elements. Wnt signaling from the nail matrix cells was necessary both to produce new nails and to make nerves grow toward the tip of the healing digit. These nerves triggered expression of FGF2, inducing the growth of bone. The skin, the nail, the nerves and the bone could all come into place and form a new fingertip.
Discussion Questions

Besides restoring missing digits, what other medical applications could come out of this discovery?

Unlike humans, normal mice can regenerate the tips of their digits throughout their lives. How might this affect efforts to apply this research to human patients?

Gout

Gout, complex disease caused by the faulty metabolism of uric acid produced in the body by breakdown of purine, a molecule found throughout all tissues and in many foods. Excessive production of uric acid or insufficient elimination of uric acid from the body by the kidneys can lead to hyperuricemia, or elevated levels of uric acid in the blood. This condition may be accompanied by the formation of crystals of uric acid in connective tissue and/or the joint space between bones. The accumulation of such crystals in the joint space can give rise to inflammation and bouts of intense pain that are the hallmark of gout.

Gout rarely affects children and young adults. It most frequently develops in adult men, especially between ages 40 and 50; it rarely strikes women before menopause. Some people may inherit a predisposition to gout; up to 20 to 25 percent of cases involve a family history of the disorder. Factors that appear to raise the risk of developing hyperuricemia or gout or may aggravate the disorder in some individuals include overweight, high alcohol consumption, excessive consumption of foods rich in purines, exposure to lead in the environment, use of certain drugs (such as diuretics, nicotinic acid, cyclosporine, and levodopa), and certain health conditions (such as untreated high blood hypertension, diabetes, high blood levels of fat and cholesterol in the blood, and arteriosclerosis).

The initial stage of the disease is marked by high blood levels of uric acid, although no symptoms appear. The buildup of uric acid crystals in joint spaces provides the basis for acute gout. Acute attacks are characterized by severe pain in the joints, most often in the big toe, but sometimes in the ankle, heel, knee, hip, shoulder, wrist, fingers, or elbow. The attack usually begins abruptly; the joint typically becomes swollen, red, inflamed, warm, and extremely tender. Untreated attacks last from a few days to a week or more. The interval between acute attacks–months or years in early stages of the disease–tends to decrease with time. Left untreated, gout may after several years progress to the advanced stage known as chronic tophacious gout. In this condition crystals of uric acid lodge as deposits of white, chalky material, or tophi, in soft body tissues and in and about the joints, where they may cause destruction of bone and bursitis. Large and deforming deposits may, after many years, settle in the outer margins of the ears, a characteristic feature of the disease. Chronic gout may also cause kidney damage, a condition called gouty nephropathy. In some people the accumulation of uric acid crystals in the kidneys leads to the formation of uric acid stones.

Treatment objectives include prevention of acute attacks, relief of the pain associated with acute attacks, avoidance of the formation of tophi and kidney stones, and prevention of gout-related disability. Acute attacks are commonly treated with orally administered nonsteroidal anti-inflammatory drugs (NSAIDs), such as indomethacin and naproxen, or with corticosteroids, given orally or injected into the affected joint or a muscle. An additional option is the alkaloid drug colchicine, which, however, may cause gastrointestinal problems in some individuals. Dietary restrictions may help reduce the severity of gout attacks; many doctors, for example, recommend limiting consumption of high-protein foods (such as meat, poultry, and fish), which can raise the level of uric acid in the blood. Chronic gout is usually treated by agents that promote excretion of uric acid, such as probenecid, and agents that inhibit production of uric acid, such as allopurinol. In some cases drug therapy proves insufficient, and it may be desirable to use surgical means to remove tophi and repair the affected joint.

Gout is sometimes confused with another inflammatory disorder–calcium pyrophosphate dihydrate deposition disease, acute attacks of which are often called pseudogout. Gout and pseudogout attacks may be characterized by similar symptoms, but their causes are different. Pseudogout results from deposits of calcium pyrophosphate dihydrate crystals, which weaken cartilage. The knees are a common site of severe attacks. Other joints that may be affected by pseudogout attacks include the wrists, shoulders, ankles, elbows, and hands.