Regenerative Medicine  

Scientists have developed a fast, efficient way to turn cells extracted from routine liposuction into liver cells, without the new cells forming tumors, as is the case with some stem cell methods.

In a feat of modern-day alchemy with huge potential for regenerative medicine, Stanford University School of Medicine scientists have developed a fast, efficient way to turn cells extracted from routine liposuction into liver cells.

The advance is described in a study published Oct. 21 in Cell Transplantation.

The scientists performed their experiments in mice, but the adipose stem cells they used came from human liposuction aspirates and became human, liver-like cells that flourished inside the mice’s bodies. This method is distinct from those producing liver cells from embryonic stem cells or induced pluripotent stem cells. Although iPS and embryonic stem cells are pluripotent — they can, in principle, differentiate into every cell type — they carry a palpable risk of forming tumors.  However, the cells produced using this new technique, which involves no intermediate pluripotent phase, show no signs of being tumorogenic.

Acute liver failure from acetaminophen alone takes about 500 lives annually and accounts for close to 60,000 emergency-room visits and more than 25,000 hospitalizations annually. Other environmental toxins, including poisonous mushrooms, contribute still more cases.

All aspects of the new fat-to-liver technique are adaptable for human use, said Gary Peltz, MD, PhD, professor of anesthesia and the study’s senior author. Creating iPS cells requires introducing foreign and potentially carcinogenic genes. But adipose stem cells merely have to be harvested from fat tissue. The process takes nine days from start to finish — fast enough to regenerate liver tissue in acute liver poisoning victims, who would otherwise die within a few weeks, barring liver transplantation.

Some 6,300 liver transplants are performed annually in the United States, with another 16,000 patients on the waiting list. Every year, more than 1,400 people die before a suitable liver can be found for them. While it can save lives, liver transplantation is complicated, risky and, even when successful, fraught with aftereffects. Typically, the recipient is consigned to a lifetime of taking immunosuppressant drugs to prevent organ rejection.

“We believe our method will be transferable to the clinic,” Peltz said. “And because the new liver tissue is derived from a person’s own cells, we do not expect that immunosuppressants will be needed.”

Liver cells are not something an adipose stem cell normally wants to turn into, Peltz said.

The Stanford team knew it was possible, though. Another way of converting liposuction-derived adipose stem cells to liver-like cells (called i-Heps, for induced hepatocytes) had been developed in 2006 by Japanese researchers. But that method, which relies on chemical stimulation, requires 30 days or longer and is inefficient; it could not produce enough material for liver reconstitution. (Working with iPS cells takes even longer; they must first be generated from adult cells before they can be converted to i-Heps.)

Using a different technique — Peltz refers to it as spherical culture — he and his associates were able to achieve the conversion within nine days with an efficiency of 37 percent, as opposed to the vastly lower yield obtained with the prior method (12 percent) or using iPS cells. (Peltz said improvements since the study’s publication now enable yields exceeding 50 percent within seven to eight days.)

Dan Xu, PhD, a postdoctoral scholar and the study’s lead author, adapted the spherical culture methodology from early embryonic-stem-cell literature. Instead of growing on flat surfaces in a laboratory dish, the harvested adipose stem cells are cultured in a liquid suspension in which they form spheroids. “This seems to make them happier,” Peltz said.

When they had enough cells, the investigators tested them by injecting them into immune-deficient laboratory mice that accept human grafts. These mice were bioengineered in 2007, in a collaboration between Peltz’s lab and study co-author Toshihiko Nishimura, MD, PhD, and other scientists at the Tokyo-based Central Institute for Experimental Animals. Only the livers of these mice contained an extra gene that would convert the antiviral compound gancyclovir into a potent toxin. When these mice were treated with gancyclovir, their liver cells died off quickly.

At this point the investigators injected 5 million i-Heps into the mice’s livers. To do that — no mean feat, as these tiny organs weigh a scant 1.8 grams — they used an ultrasound-guided injection procedure that is routinely employed in gastroenterological clinics for biopsies.

Four weeks later, the investigators examined the mice’s blood and found the presence of a protein (human serum albumin) that is only produced by human liver cells and was shown to be an accurate proxy for the number of new human liver cells in these experimental mice’s livers. The mice’s blood had substantial human serum albumin levels, which nearly tripled in the following four weeks. These blood levels correspond with the repopulation of roughly 10-20 percent of the mice’s pre-destroyed livers by new human liver tissue. (Past studies have shown only miniscule human serum albumin production, at best, in mice given similar amounts of chemically induced i-Heps.)

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Blood tests also revealed that the mice’s new liver tissue was discharging its waste-filtration responsibility. Examination of the livers themselves showed that the transplanted cells had integrated into the liver, expressed surface markers unique to mature human hepatocytes and produced multi-cell structures required for human bile duct formation. Other tests indicated that the spherically cultured i-Heps resembled natural human hepatocytes more closely than did i-Heps produced from iPS cells.

Importantly, two months after injection of i-Heps produced by spherical culture, there was no evidence of tumor formation. But mice in which IPS-cell-originated i-Heps were introduced developed multiple tumors, which could be felt through the body surface within three weeks.

“To be successful, we must regenerate about half of the damaged liver’s original cell count,” said Peltz. With spherical culture, he said, close to a billion injectable iHeps can be produced from 1 liter of liposuction aspirate, readily obtained from a single liposuction procedure. The cell replication that takes place after injection expands that number further, to over 100 billion i-Heps.

That could be enough to substitute for a human liver transplant, Peltz said. Stanford’s Office of Technology Licensing has filed a patent on the use of spherical culture for hepatocyte induction. Peltz’s group is optimizing the culture and injection techniques, talking to the U.S. Food and Drug Administration, and gearing up for safety tests on large animals. Barring setbacks, the new method could be ready for clinical trials within two to three years, he estimated.

SOURCE  Stanford University School of Medicine

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 Mind Reading  

Researchers have found the first solid evidence that the pattern of brain activity seen in someone performing a mathematical exercise under experimentally controlled conditions is very similar to that observed when the person engages in thinking about numbers and quantities in general.

A brain region activated when people are asked to perform mathematical calculations in an experimental setting is similarly activated when they use numbers — or even imprecise quantitative terms, such as “more than”— in everyday conversation, according to a study by Stanford University School of Medicine scientists.

Using a new method, the researchers collected the first solid evidence that the pattern of brain activity seen in someone performing a mathematical exercise under experimentally controlled conditions is very similar to that observed when the person engages in quantitative thought in the course of daily life.

“We’re now able to eavesdrop on the brain in real life,” said Josef Parvizi, MD, PhD, associate professor of neurology and neurological sciences and director of Stanford’s Human Intracranial Cognitive Electrophysiology Program. Parvizi is the senior author of the study, published in Nature Communications. The study’s lead authors are postdoctoral scholar Mohammad Dastjerdi, MD, PhD, and graduate student Muge Ozker.

The finding could lead to “mind-reading” applications that, for example, would allow a patient who is rendered mute by a stroke to communicate via passive thinking. Conceivably, it could also lead to more dystopian outcomes: chip implants that spy on or even control people’s thoughts.

“This is exciting, and a little scary,” said Henry Greely, JD, the Deane F. and Kate Edelman Johnson Professor of Law and steering committee chair of the Stanford Center for Biomedical Ethics, who played no role in the study but is familiar with its contents and described himself as “very impressed” by the findings. “It demonstrates, first, that we can see when someone’s dealing with numbers and, second, that we may conceivably someday be able to manipulate the brain to affect how someone deals with numbers.”

The researchers monitored electrical activity in a region of the brain called the intraparietal sulcus, known to be important in attention and eye and hand motion. Previous studies have hinted that some nerve-cell clusters in this area are also involved in numerosity, the mathematical equivalent of literacy.

However, the techniques that previous studies have used, such as functional magnetic resonance imaging, are limited in their ability to study brain activity in real-life settings and to pinpoint the precise timing of nerve cells’ firing patterns. These studies have focused on testing just one specific function in one specific brain region, and have tried to eliminate or otherwise account for every possible confounding factor. In addition, the experimental subjects would have to lie more or less motionless inside a dark, tubular chamber whose silence would be punctuated by constant, loud, mechanical, banging noises while images flashed on a computer screen.

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“This is not real life,” said Parvizi. “You’re not in your room, having a cup of tea and experiencing life’s events spontaneously.” A profoundly important question, he said, is: “How does a population of nerve cells that has been shown experimentally to be important in a particular function work in real life?”

His team’s method, called intracranial recording, provided exquisite anatomical and temporal precision and allowed the scientists to monitor brain activity when people were immersed in real-life situations. Parvizi and his associates tapped into the brains of three volunteers who were being evaluated for possible surgical treatment of their recurring, drug-resistant epileptic seizures.

The procedure involves temporarily removing a portion of a patient’s skull and positioning packets of electrodes against the exposed brain surface. For up to a week, patients remain hooked up to the monitoring apparatus while the electrodes pick up electrical activity within the brain. This monitoring continues uninterrupted for patients’ entire hospital stay, capturing their inevitable repeated seizures and enabling neurologists to determine the exact spot in each patient’s brain where the seizures are originating.

During this whole time, patients remain tethered to the monitoring apparatus and mostly confined to their beds. But otherwise, except for the typical intrusions of a hospital setting, they are comfortable, free of pain and free to eat, drink, think, talk to friends and family in person or on the phone, or watch videos.

The electrodes implanted in patients’ heads are like wiretaps, each eavesdropping on a population of several hundred thousand nerve cells and reporting back to a computer.

In the study, participants’ actions were also monitored by video cameras throughout their stay. This allowed the researchers later to correlate patients’ voluntary activities in a real-life setting with nerve-cell behavior in the monitored brain region.

As part of the study, volunteers answered true/false questions that popped up on a laptop screen, one after another. Some questions required calculation — for instance, is it true or false that 2+4=5? — while others demanded what scientists call episodic memory — true or false: I had coffee at breakfast this morning. In other instances, patients were simply asked to stare at the crosshairs at the center of an otherwise blank screen to capture the brain’s so-called “resting state.”

Consistent with other studies, Parvizi’s team found that electrical activity in a particular group of nerve cells in the intraparietal sulcus spiked when, and only when, volunteers were performing calculations.

Afterward, Parvizi and his colleagues analyzed each volunteer’s daily electrode record, identified many spikes in intraparietal-sulcus activity that occurred outside experimental settings, and turned to the recorded video footage to see exactly what the volunteer had been doing when such spikes occurred.

They found that when a patient mentioned a number — or even a quantitative reference, such as “some more,” “many” or “bigger than the other one” — there was a spike of electrical activity in the same nerve-cell population of the intraparietal sulcus that was activated when the patient was doing calculations under experimental conditions.

That was an unexpected finding. “We found that this region is activated not only when reading numbers or thinking about them, but also when patients were referring more obliquely to quantities,” said Parvizi.

“These nerve cells are not firing chaotically,” he said. “They’re very specialized, active only when the subject starts thinking about numbers. When the subject is reminiscing, laughing or talking, they’re not activated.” Thus, it was possible to know, simply by consulting the electronic record of participants’ brain activity, whether they were engaged in quantitative thought during nonexperimental conditions.

Any fears of impending mind control are, at a minimum, premature, said Greely. “Practically speaking, it’s not the simplest thing in the world to go around implanting electrodes in people’s brains. It will not be done tomorrow, or easily, or surreptitiously.”

Parvizi agreed. “We’re still in early days with this,” he said. “If this is a baseball game, we’re not even in the first inning. We just got a ticket to enter the stadium.”

SOURCE  Stanford School of Medicine

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