Cardiac muscle cells derived from human embryonic stem cells

The promise of embryonic stem cells lies in their ability to develop into any type of cell in the human body, which should allow us to replace tissues lost due to injury or disease. But it's one thing to generate replacement cells; it's another thing to generate entire tissues and integrate them into a functioning organ. A paper released by Nature now reports some success with turning human embryonic stem cells (hESCs) into cardiac cells and getting them to beat in synchrony with a damaged heart.

The blockage of blood vessels in the heart, either through clots or occlusion, causes the cells that rely on the blocked vessel to die off. This both weakens the heart structurally and changes the ability of the heart to beat in an organized manner, since the scar tissue that develops doesn't conduct electrical impulses. Serious arrhythmias can develop as a result of this changed activity, and these can sometimes end up causing the heartbeat to be lost entirely.

Embryonic stem cells have been used to try to repair damaged hearts for a while, starting with simple experiments where the stem cells themselves were injected. More recently, researchers have induced hESCs to form cardiac muscle cells (cardiomyocytes) before implanting them in a damaged heart (typically that of a mouse or rat). This treatment tends to increase the ability of the heart to pump blood, indicating that stem cells can reverse the weakening of the heart.

But it has been harder to get at the electrical integration of these stem cells, in part because the rodents that the researchers used have a very fast heartbeaton the order of 400-600 beats a minute. (The human heart rate is normally under 100 beats per minute.) So, the new work relied on the guinea pig, which apparently has a heart rate that is only about 200-250 beats per minute.

The authors took an hESC line and induced it to form cardiomyocytes, which were injected into injured hearts and then allowed to integrate with the injured heart for a while. Rather than focusing on blood flow, the authors tracked the development of arrhythmias. It turns out that the hESC-derived cardiomyocytes suppressed them. The guinea pigs treated with them had the lowest rate of premature ventricular contractions, or PVCs, which occur when the lower chambers of the heart beat ahead of schedule. They also went into tachycardia, or a run of rapid heartbeats, less often.

To track the behavior of the hESC-derived cardiac cells, the authors inserted a gene for a protein that becomes fluorescent in response to changes in calcium, which accompany the electrical impulses that drive a heartbeat. By tracking whether a cell was glowing, the authors could determine whether the human cells were tied to the regular guinea pig heartbeat.

Here, the results were a bit mixed. In areas where the hESC-derived cells were stuck in an area with lots of scar tissue, they tended to contract on their own, without significant influence from the guinea pig's rhythm. But in other areas where the cells were clear of nearby scar tissue, they tended to tie in nicely with the heart's overall rhythmeven when they weren't necessarily close to any guinea pig tissue.

The results are very promising, in that they show that embryonic stem cells can be used to create a large population of cardiomyocytes that can then function normally when placed back into a heart. But they also make it clear that scar tissue remains a problem in damaged hearts. Even if muscle tissue gets replaced, it won't integrate well if there's a significant amount of scar tissue around. This provides researchers with an obvious target for future efforts.

Incidentally, a number of the researchers involved in this work were based at US institutions. Early in the history of stem cell research, legislation was considered that would ban the creation of human-animal hybrids. Although it was probably written with Frankenstein-like chimeras in mind, some of it was so broadly worded that it would have banned basic safety and efficacy research such as the work described by this paper. Fortunately, it never passed, so US researchers are still able to contribute to work like this.

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Heart tissue derived from embryonic stem cells doesn't skip a beat

Shinya Yamanaka aims to produce cell lines from fetal blood cells.

M. Naka/Aflo/Newscom

Progress toward stem-cell therapies has been frustratingly slow, delayed by research challenges, ethical and legal barriers and corporate jitters. Now, stem-cell pioneer Shinya Yamanaka of Kyoto University in Japan plans to jump-start the field by building up a bank of stem cells for therapeutic use. The bank would store dozens of lines of induced pluripotent stem (iPS) cells, putting Japan in an unfamiliar position: at the forefront of efforts to introduce a pioneering biomedical technology.

A long-held dream of Yamanakas, the iPS Cell Stock project received a boost last month, when a Japanese health-ministry committee decided to allow the creation of cell lines from the thousands of samples of fetal umbilical-cord blood held around the country. Yamanakas plan to store the cells for use in medicine is a bold move, says George Daley, a stem-cell biologist at Harvard Medical School in Boston, Massachusetts. But some researchers question whether iPS cells are ready for the clinic.

Yamanaka was the first researcher to show, in 2006, that mature mouse skin cells could be prodded into reverting to stem cells1 capable of forming all bodily tissues. The experiment, which he repeated2 with human cells in 2007, could bypass ethical issues associated with stem cells derived from embryos, and the cells could be tailor-made to match each patient, thereby avoiding rejection by the immune system.

Japan is pumping tens of millions of dollars every year into eight long-term projects to translate iPS cell therapies to the clinic, including a US$2.5-million-per-year effort to relieve Parkinsons disease at Kyoto Universitys Center for iPS Cell Research and Application (CiRA), which Yamanaka directs. That programme is at least three years away from clinical trials. The first human clinical trials using iPS cells, an effort to repair diseased retinas, are planned for next year at the RIKEN Center for Developmental Biology in Kobe.

Those trials will not use cells from Yamanakas Stock. But if they or any other iPS cell trials succeed, demand for the cells will explode, creating a supply challenge. Deriving and testing iPS cells tailored to individual patients could take six months for each cell line and cost tens of thousands of dollars.

Yamanakas plan is to create, by 2020, a standard array of 75 iPS cell lines that are a good enough match to be tolerated by 80% of the population. To do that, Yamanaka needs to find donors who have two identical copies of each of three key genes that code for immune-related cell-surface proteins called human leukocyte antigens (HLAs). He calculates that he will have to sift through samples from some 64,000 people to find 75 suitable donors.

Using blood from Japans eight cord-blood banks will make that easier. The banks hold some 29,000samples, all HLA-characterized, and Yamanaka is negotiating to gain access to those that prove unusable for other medical procedures. One issue remains unresolved: whether the banks need to seek further informed consent from donors, most of whom gave the blood under the understanding that it would be used for treating or studying leukaemia. Each bank will determine for itself whether further consent is needed.

Yamanaka has already built a cell-processing facility on the second floor of CiRA and is now applying for ethics approval from Kyoto University to create the stock. Takafumi Kimura, a CiRA biologist and head of the projects HLA analysis unit, says that the team hopes to derive the first line, carrying a set of HLA proteins that matches that of 8% of Japans population, by next March.

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Stem-cell pioneer banks on future therapies

--Studies in mice reveal how mood-altering drugs may affect brain stem cells

Newswise Working with mice, Johns Hopkins researchers say they have figured out how stem cells found in a part of the brain responsible for learning, memory and mood regulation decide to remain dormant or create new brain cells. Apparently, the stem cells listen in on the chemical communication among nearby neurons to get an idea about what is stressing the system and when they need to act.

The researchers say understanding this process of chemical signaling may shed light on how the brain reacts to its environment and how current antidepressants work, because in animals these drugs have been shown to increase the number of brain cells. The findings are reported July 29 in the advance online publication of Nature.

What we learned is that brain stem cells dont communicate in the official way that neurons do, through synapses or by directly signaling each other, says Hongjun Song, Ph.D., professor of neurology and director of Johns Hopkins Medicines Institute for Cell Engineerings Stem Cell Program. Synapses, like cell phones, allow nerve cells to talk with each other. Stem cells dont have synapses, but our experiments show they indirectly hear the neurons talking to each other; its like listening to someone near you talking on a phone.

The indirect talk that the stem cells detect is comprised of chemical messaging fueled by the output of neurotransmitters that leak from neuronal synapses, the structures at the ends of brain cells that facilitate communication. These neurotransmitters, released from one neuron and detected by a another one, trigger receiving neurons to change their electrical charges, which either causes the neuron to fire off an electrical pulse propagating communication or to settle down, squelching further messages.

To find out which neurotransmitter brain stem cells can detect, the researchers took mouse brain tissue, attached electrodes to the stem cells and measured any change in electrical charge after the addition of certain neurotransmitters. When they treated the stem cells with the neurotransmitter GABA a known signal-inhibiting product the stem cells electrical charges changed, suggesting that the stem cells can detect GABA messages.

To find out what message GABA imparts to brain stem cells, the scientists used a genetic trick to remove the gene for the GABA receptor the protein on the surface of the cell that detects GABA only from the brain stem cells. Microscopic observation of brain stem cells lacking the GABA receptor over five days showed these cells replicated themselves, or produced glial cells support cells for the neurons in the brain. Brain stem cells with their GABA receptors intact appeared to stay the same, not making more cells.

Next, the team treated normal mice with valium, often used as an anti-anxiety drug and known to act like GABA by activating GABA receptors when it comes in contact with them. The scientists checked the mice on the second and seventh day of valium use and counted the number of brain stem cells in untreated mice and mice treated with the GABA activator. They found the treated mice had many more dormant stem cells than the untreated mice.

Traditionally GABA tells neurons to shut down and not continue to propagate a message to other neurons, says Song. In this case the neurotransmitter also shuts off the stem cells and keeps them dormant.

The brain stem cell population in mice (and other mammals, including humans) is surrounded by as many as 10 different kinds of intermingled neurons, says Song, and any number of these may be keeping stem cells dormant. To find out which neurons control the stem cells, the researchers inserted special light-activating proteins into the neurons that trigger the cells to send an electrical pulse, as well as to release neurotransmitter, when light shines on them. By shining light to activate a specific type of neuron and monitoring the stem cells with an electrode, Songs team showed that one of the three types of neurons tested transmitted a signal to the stem cells causing a change in electrical charge in the stem cells. The neurons messaging the stem cells are parvalbumin-expressing interneurons.

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Brain's Stem Cells "Eavesdrop" to Find Out When to Act

ANN ARBOR In the first human study of its kind, researchers found that using stem cells to re-grow craniofacial tissues mainly bone proved quicker, more effective and less invasive than traditional bone regeneration treatments.

Researchers from the University of Michigan School of Dentistry and the Michigan Center for Oral Health Research partnered with Ann Arbor-based Aastrom Biosciences Inc. in the clinical trial, which involved 24 patients who required jawbone reconstruction after tooth removal.

Patients either received experimental tissue repair cells or traditional guided bone regeneration therapy. The tissue repair cells, called ixmyelocel-T, are under development at Aastrom, which is a UM spinout company.

For a video of the procedure, see: http://youtu.be/lWu_DEJfZVk

In patients with jawbone deficiencies who also have missing teeth, it is very difficult to replace the missing teeth so that they look and function naturally, said Darnell Kaigler, principal investigator and assistant professor at the UM School of Dentistry. This technology and approach could potentially be used to restore areas of bone loss so that missing teeth can be replaced with dental implants.

William Giannobile, director of the Michigan Center for Oral Health Research and chair of the UM Department of Periodontics and Oral Medicine, is co-principal investigator on the project.

The treatment is best suited for large defects such as those resulting from trauma, diseases or birth defects, Kaigler said. These defects are very complex because they involve several different tissue types bone, skin, gum tissue and are very challenging to treat.

The main advantage to the stem cell therapy is that it uses the patients own cells to regenerate tissues, rather than introducing man-made, foreign materials, Kaigler said.

The results were promising. At six and 12 weeks following the experimental cell therapy treatment, patients in the study received dental implants. Patients who received tissue repair cells had greater bone density and quicker bone repair than those who received traditional guided bone regeneration therapy.

In addition, the experimental group needed less secondary bone grafting when getting their implants.

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Stem Cell Therapy Could Offer New Hope For Head, Mouth Injuries, Defects

When transplanted into guinea pig hearts, human heart muscle cells (pictured) can beat in time with resident cells.

MEDIMAGE / SPL

Damaged skin and liver can often repair themselves, but the heart rarely heals well and heart disease is the world's leading cause of death. Research published today raises hopes for cell therapies, showing that heart muscle cells differentiated from human embryonic stem cells can integrate into existing heart muscle[1].

What we have done is prove that these cells do what working heart muscles do, which is beat in sync with the rest of the heart, says Chuck Murry, a cardiovascular biologist at the University of Washington in Seattle, who co-led the research.

It has been difficult to assess cell therapies in animal models because human cells cannot keep up with the heart rates of some small rodents. Cardiomyocytes derived from human embryonic stem (ES) cells typically beat fewer than 150 times a minute. External electrical stimulation can increase that rate, but only up to about 240 beats per minute, says Michael LaFlamme, a cardiovascular biologist at the University of Washington and the other co-leader on the project. Rats and mice have heart rates of around 400 and 600 beats per minute, respectively.

However, guinea pigs have a heart rate of 200250 beats per minute, near the limit for human cardiomyocytes. After working out ways to suppress guinea pigs immune systems so that they would accept human cells, Murry, LaFlamme and their co-workers began transplantation experiments. They also devised a way to make assessing electrical activity straightforward: using recent genetic-engineering technology, they inserted a sensor gene into the human ES cells so that cardiomyocytes derived from them would fluoresce when they contracted.

From the first experiment with the sensor in guinea pigs, it was obvious that the transplanted cells were beating in time with the rest of the heart, says LaFlamme. When he looked into the chest cavity, the heart was flashing back at us, he says.

The human cells seemed to aid healing: four weeks after the researchers killed regions of the guinea pigs hearts to simulate a heart attack, the hearts of animals that received cardiomyocytes exhibited stronger contractions than those that received other cell types. And cardiomyocyte transplants did not seem to cause irregular heartbeats, a common concern for cell-replacement therapy in the heart. In fact, the transplants seemed to suppress arrhythmias.

But it will be a long road from demonstrating this sort of integration to demonstrating possible therapeutic benefits, says Glenn Fishman, a cardiologist at New York University Langone School of Medicine, who was not involved in the work. The conclusion that the human cells can connect with the guinea pig tissue is true, he says, but the clinical implications are a bit of a stretch.

Cardiomyocytes engrafted in only a tiny percentage of scar tissue, Fishman explains, and the area seems too small to produce much additional pumping force. He suspects that the benefits seen stem from the 'paracrine effect', in which transplanted cells secrete factors that rejuvenate damaged host tissue. In fact, many researchers are exploring such strategies to prompt damaged heart tissue to restore itself, he says.

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Guinea pig hearts beat with human cells