cardiomyocytes

Stem cell stories that caught our eye: bubble baby therapy a go in UK, in-utero stem cell trial and novel heart disease target

/ Todd Dubnicoff / Leave a comment

There were lots of CIRM mentions in the news this week. Here are two brief recaps written by Karen Ring to get you up to speed. A third story by Todd Dubnicoff summarizes an promising finding related to heart disease by researchers in Singapore.  

CIRM-funded “bubble baby” disease therapy gets special designation by UK.
Orchard Therapeutics, a company based in the UK and the US, is developing a stem cell-based gene therapy called OTL-101 to treat a primary immune disease called adenosine-deaminase deficient severe combined immunodeficiency (ADA-SCID), also known as “bubble baby disease”. CIRM is funding a Phase 1/2 clinical trial led by Don Kohn of UCLA in collaboration with Orchard and the University College in London.

In July, the US Food and Drug Administration (FDA) awarded OTL-101 Rare Pediatric Disease Designation (read more about it here), which makes the therapy eligible for priority review by the FDA, and could give it a faster route to being made more widely available to children in need.

On Tuesday, Orchard announced further good news that OTL-101 received “Promising Innovative Medicine Designation” by the UK’s Medicines and Healthcare Products Regulatory Agency (MHRA). In a news release, the company explained how this designation bodes well for advancing OTL-101 from clinical trials into patients,

“The designation as Promising Innovative Medicine is the first step of a two-step process under which OTL-101 can benefit from the Early Access to Medicine Scheme (“EAMS”). Nicolas Koebel, Senior Vice President for Business Operations at Orchard, added: “With this PIM designation we can potentially make OTL-101 available to UK patients sooner under the Early Access to Medicine Scheme”.

CIRM funded UCSF clinical trial mentioned in SF Business Times
Ron Leuty, reporter at the San Francisco Business Times, published an article about a CIRM-funded trial out of UCSF that is targeting a rare genetic blood disease called alpha thalassemia major, describing it as, “The world’s first in-utero blood stem cell transplant, soon to be performed at the University of California, San Francisco, could point the way toward pre-birth cures for a range of blood diseases, such as sickle cell disease.”

Alpha Thalassemia affects the ability of red blood cells to carry oxygen because of a reduction in a protein called hemoglobin. The UCSF trial, spearheaded by UCSF Pediatric surgeon Dr. Tippi MacKenzie, is hoping to use stem cells from the mother to treat babies in the womb to give them a better chance at surviving after birth.

In an interview with Leuty, Tippi explained,

“Our goal is to put in enough cells so the baby won’t need another transplant. But even if we fall short, if we can just establish 1 percent maternal cells circulating in the child, it will establish tolerance and then they can get the booster transplant.”

She also emphasized the key role that CIRM funded played in the development and launch of this clinical trial.

“CIRM is about more than funding for studies, MacKenzie said. Agency staff has provided advice about how to translate animal studies into work in humans, she said, as well as hiring an FDA consultant, writing an investigational new drug application and setting up a clinical protocol.”

“I’m a clinician, but running a clinical trial is different,” MacKenzie said. “CIRM’s been incredibly helpful in helping me navigate that.”

Heart, heal thyself: the story of Singheart
When you cut your finger or scrape a knee, a scab forms, allowing the skin underneath to regenerate and repair itself. The heart is not so lucky – it has very limited self-healing abilities. Instead, heart muscle cells damaged after a heart attack form scar tissue, making each heart beat less efficient. This condition can lead to chronic heart disease, the number one killer of both men and women in the US.

A mouse heart cell with 2 nuclei (blue) and Singheart RNA labelled by red fluorescent dyes.
Credit: A*STAR’s Genome Institute of Singapore

Research has shown that newborn mice retain the ability to completely regenerate and repair injuries to the heart because their heart muscle cells, or cardiomyocytes, are still able to divide and replenish damaged cells. But by adulthood, the mouse cardiomyocytes lose the ability to stimulate the necessary cell division processes. A research team in Singapore wondered what was preventing cardiomyocytes cell division in adult mice and if there was some way to lift that block.

This week in Nature Communications, they describe the identification of a molecule they call Singheart that may be the answer to their questions. Using tools that allow the analysis of gene activity in single cells revealed that a rare population of diseased cardiomyocytes are able to crank up genes related to cell division. And further analysis showed Singheart, a specialized genetic molecule called a long non-coding RNA, played a role in blocking this cell division gene.

As lead author Dr. Roger Foo, a principal investigator at Genome Institute of Singapore (GIS) and the National University Health System (NUHS), explained in a press release, these findings may lead to new self-healing strategies for heart disease,

“There has always been a suspicion that the heart holds the key to its own healing, regenerative and repair capability. But that ability seems to become blocked as soon as the heart is past its developmental stage. Our findings point to this potential block that when lifted, may allow the heart to heal itself.”

Understanding two heart problems by studying the domino effect of one gene network

/ Todd Dubnicoff / Leave a comment

Although heart muscle cells, or cardiomyocytes, are specialized to help pump blood to the organs, they nonetheless carry all the genetic instructions for becoming a nerve cell, an intestinal cell, a liver or any cell type in the body. But at the moment in time that the fetal heart begins to develop, master switch proteins, called transcription factors, act like the first tile in an extremely complex pattern of dominos and set off a chain of events which lead to the activation of heart muscle specific genes in cardiomyocytes as well as the silencing of genes important for the development other cells types.

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cardiomyocytes

It’s truly amazing that this process comes together to create functioning hearts in the about 355,000 babies that are born in the world each day. But it isn’t always flawless as heart defects occur in about 1% of all live births. By studying a family with a history of heart defects, scientists at the Gladstone Institutes have gained a deeper understanding of how gene networks go awry,  causing heart defects as well as heart disease later in life. This CIRM-funded work was published today in Cell.

Half the children in the family studied by the Gladstone team were born with a hole in the wall between the two chambers of the heart. Back in 2003, the family approached Deepak Srivastava, head of the cardiovascular institute at Gladstone, for help. A genetic analysis by Srivastava’s team found that all of the affected children carried a mutation in the GATA4 gene, which encodes a heart specific transcription factor protein. Seven years later the children developed heart disease that led to weaker heart pumping. Although the two heart problems were not related, they suspected both were caused by the GATA4 mutation and sought to understand how that could be the case.

Srivastava’s team sought to understand how the GATA4 mutation could be causing both health problems. They collected skin samples from the affected children and generated cardiomyocytes using the induced pluripotent stem cell technique. Cells were also collected from the children’s healthy siblings. In the laboratory, the cells were analyzed for how well they functioned, such as their ability to contract. All of these tests showed that the cells carrying the GATA4 mutation had impaired function compared to the healthy cells. These findings provide a basis for the heart disease found in the children during their teens.

In terms of the heart wall defect, the team examined the GATA4 protein’s interaction with the protein TBX5, another transcription factor that is also mutated in cases of this defect. Both proteins regulate genes by directly binding to DNA as well as interacting with each other. In cells with the defective GATA4, the research discovered TBX5 did not bind well to the DNA. The lack of TBX5 led to a disruption in the activation of genes that play a role in the development of the heart wall.

TBX5 and GATA4 also work together in cardiomyocytes to silence genes that play a role in other cell types. But the scientists found that the because the GATA4 mutation hindered its interaction with TBX5, those non-heart specific genes we’re no longer repressed causing further disruption to proper cardiomyocyte development. Srivastava summed up these results in an institute press release:

srivastava-profile

Deepak Srivastava

“By studying the patients’ heart cells in a dish, we were able to figure out why their hearts were not pumping properly. Investigating their genetic mutation revealed a whole network of genes that went awry, first causing septal [heart wall] defects and then the heart muscle dysfunction.”

Now, because GATA4 and TBX5 are those first domino tiles in very intricate networks of genes, targeting those proteins for future therapy development wouldn’t be wise. Their effects are so widespread that blocking their actions would do more harm than good. But finding drugs that might affect only a branch of GATA4/TBX5 actions could result in new therapy approaches to heart defects and disease.

deepak-yen-sin-22 Deepak Srivastava and Yen-Sin Ang [Photo: Chris Goodfellow, Gladstone Institutes]

Yen-Sin Ang, the first author on the report, thinks these finding could prove fruitful for other diseases as well:

“It’s amazing that by studying genes in a two-dimensional cluster of heart cells, we were able to discover insights into a disease that affects a complicated three-dimensional organ. We think this conceptual framework could be used to study other diseases caused by mutations in proteins that serve as master regulators of whole gene networks.”

Using skin cells to repair damaged hearts

/ Kevin McCormack / Leave a comment
heart-muscle

Heart muscle  cells derived from skin cells

When someone has a heart attack, getting treatment quickly can mean the difference between life and death. Every minute delay in getting help means more heart cells die, and that can have profound consequences. One study found that heart attack patients who underwent surgery to re-open blocked arteries within 60 minutes of arriving in the emergency room had a six times greater survival rate than people who had to wait more than 90 minutes for the same treatment.

Clearly a quick intervention can be life-saving, which means an approach that uses a patient’s own stem cells to treat a heart attack won’t work. It simply takes too long to harvest the healthy heart cells, grow them in the lab, and re-inject them into the patient. By then the damage is done.

Now a new study shows that an off-the-shelf approach, using donor stem cells, might be the most effective way to go. Scientists at Shinshu University in Japan, used heart muscle stem cells from one monkey, to repair the damaged hearts of five other monkeys.

In the study, published in the journal Nature, the researchers took skin cells from a macaque monkey, turned those cells into induced pluripotent stem cells (iPSCs), and then turned those cells into cardiomyocytes or heart muscle cells. They then transplanted those cardiomyocytes into five other monkeys who had experienced an induced heart attack.

After 3 months the transplanted monkeys showed no signs of rejection and their hearts showed improved ability to contract, meaning they were pumping blood around the body more powerfully and efficiently than before they got the cardiomyocytes.

It’s an encouraging sign but it comes with a few caveats. One is that the monkeys used were all chosen to be as close a genetic match to the donor monkey as possible. This reduced the risk that the animals would reject the transplanted cells. But when it comes to treating people, it may not be feasible to have a wide selection of heart stem cell therapies on hand at every emergency room to make sure they are a good genetic match to the patient.

The second caveat is that all the transplanted monkeys experienced an increase in arrhythmias or irregular heartbeats. However, Yuji Shiba, one of the researchers, told the website ResearchGate that he didn’t think this was a serious issue:

“Ventricular arrhythmia was induced by the transplantation, typically within the first four weeks. However, this post-transplant arrhythmia seems to be transient and non-lethal. All five recipients of [the stem cells] survived without any abnormal behaviour for 12 weeks, even during the arrhythmia. So I think we can manage this side effect in clinic.”

Even with the caveats, this study demonstrates the potential for a donor-based stem cell therapy to treat heart attacks. This supports an approach already being tested by Capricor in a CIRM-funded clinical trial. In this trial the company is using donor cells, derived from heart stem cells, to treat patients who developed heart failure after a heart attack. In early studies the cells appear to reduce scar tissue on the heart, promote blood vessel growth and improve heart function.

The study from Japan shows the possibilities of using a ready-made stem cell approach to helping repair damage caused by a heart attacks. We’re hoping Capricor will take it from a possibility, and turn it into a reality.

If you would like to read some recent blog posts about Capricor go here and here.

Easier, Cheaper Stem Cell-Based Heart Muscle Sets Stage for Large-Scale Drug Development

/ Todd Dubnicoff / Leave a comment

The great inventions – like the automobile, the Internet or aviation – are marked as important turning points in human history. But it’s usually the additional tinkering that goes on in the ensuing years after the initial invention that makes the technology feasible in terms of cost, reproducibility and mass production.

The same holds true for the Nobel prize winning induced pluripotent stem cell (iPS) technique. The sight of human iPS-derived heart muscle cells, or cardiomyocytes, beating in a petri dish brought a lot of early excitement in the late 2000’s (and still does today) about the potential of using human cells rather than animal models to screen for novel heart disease therapies and to test for drug toxicity.

And since iPS cells can be created directly from the skin or blood of a heart disease patient, it opened new opportunities to better understand the cellular basis of heart disease.

The Earth isn’t flat and neither is the heart
But the human heart is more than a two dimensional layer of cardiomyocytes in a petri dish. As a result, more complex three dimensional miniature heart structures that better mimic cardiac function have been developed over time. Now a CIRM-funded research team at the Gladstone Institutes has gone a step further and devised a “Micro-Heart Muscle” (µHM) technique that is easy to make and uses much fewer cells. The method detailed in Scientific Reports yesterday is poised to make large-scale, high-throughput drug and toxicology testing for heart disease therapies a reality.

Prior to this current publication, the Engineered Heart Muscle (EHM) has been the gold standard for petri dish models of human heart function. To better reflect the cellular environment of heart tissue than a simple layer of heart cells, EHM is composed of a mixture of iPS-derived cardiomyocytes, fibroblasts, and extracellular matrix, a natural scaffold that supports the heart’s cellular structure. These components are grown into 3D molds in a lab dish and embedded into posts that give the muscle cells something to contract against. This setup provides a means to do detailed analysis of the impact of drugs on heart muscle function. While these miniature EHM structures successfully produce physiologically relevant heart tissue for testing in the lab, they nonetheless carry some practical limitations. The complexity of the molds and the need for millions of cells for each EHM tissue makes the cost of large-scale drug development experiments too high. And the use of extracellular matrix would muck up the miniaturized instrumentation that is used for these therapy development efforts.

Dog bones: the key to easier, cheaper muscle tissue
The Gladstone team, led by Bruce Conklin, has overcome these challenges with their Micro-Heart Muscle product. With some educated trial and error, they zeroed in on a simple dog bone shaped mold to grow the mixture of cells in. The shape of the mold encouraged the muscle cells to self organize and grow into contracting tissue without the need of extracellular matrix. And better yet, less than 10,000 cells were needed to form each tissue. Here’s a cool video, recorded by first author Nathaniel Huebsch, of the cells in action:

These technical improvements of the Micro-Heart Muscle could help make large-scale, systematic approaches to study heart disease and toxicology a reality. As you read Conklin’s summary of the results in a Gladstone press release, you can hear his excitement about the future applications of this method:

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Bruce Conklin, Gladstone Institutes Senior Investigator

“The beauty of this technique is that it is very easy and robust, but it still allows you to create three-dimensional miniature tissues that function like normal tissues. Our research shows that you can create these complex tissues with a simple template that exploits the inherent properties of these cells to self-organize. We think that the micro heart muscle will provide a superior resource for conducting research and developing therapies for heart disease.”

 

Micro-Heart Muscle: the Model T of iPS innovations?
Karl Benz is generally credited as the inventor of the automobile, but a few years later it was Henry Ford’s efficient assembly line and manufacturing process that helped make mass production and affordability of the car possible. In a somewhat similar way, Shinya Yamanaka’s iPS technique will no doubt go down as one of the greatest inventions in the 21st century and maybe Conklin’s team’s Micro-Heart Muscle will also feature prominently in the history books as a follow up innovation that made the development of heart disease therapies possible.

Bruce-&-Nate-'web

Senior author Bruce Conklin and first author Nathaniel Huebsch [Photo: Chris Goodfellow]

A cardiac love triangle: how transcription factors interact to make a heart

/ Karen Ring / Leave a comment

 Here’s a heartfelt science story for all those Valentine’s day fans out there. Scientists from the Gladstone Institutes have identified how a group of transcription factors interact during embryonic development to make a healthy heart. Their work will increase our biological understanding of how the heart is formed and could produce new methods for treating cardiovascular disease.

The study, published today in the journal Cell, describes a tumultuous love story between cardiac transcription factors. Transcription factors are proteins that orchestrate gene expression. They have the power to turn genes on or off by binding to specific DNA sequences and recruiting other proteins that will eventually turn the information encoded in that gene into a functional protein.

Every organ has its own special group of transcription factors that coordinate the gene expression required for that organ’s development. Often times, transcription factors within a group directly interact with each other and work together to conduct a specific sequence of events. These interactions are essential for making healthy tissues and organs, but scientists don’t always understand how these interactions work.

For the heart, scientists have already identified a group of transcription factors essential for cardiac development, and genetic mutations in any of these factors can impair heart formation and cause heart defects in newborns. What’s not known, however, are the details on how some of these cardiac transcription factors interact to get their job done.

A cardiac love triangle

In the Gladstone study, the scientists focused on how three key cardiac transcription factors – NKX2.5, TBX5, and GATA4 – interact during heart development. They first proved that these transcription factors are essential for the formation of the heart in mouse embryos. When they eliminated the presence of one of the three factors from the developing mouse embryo, they observed abnormal heart development and heart defects. When they removed two factors (NKX2.5 and TBX5), the results were even worse – the heart wasn’t able to form and none of the embryos survived.

Normal heart muscle cells, courtesy Kyoto University

Normal cardiomyocytes or heart cells, courtesy Kyoto University

Next, they studied how these transcription factors interact to coordinate gene expression in heart cells called cardiomyocytes made from mouse embryonic stem cells that lacked either NKX2.5, TBX5, or both of these factors. Compared to normal heart cells, cardiomyocytes that lacked one or both of these two transcription factors started beating at inappropriate times – either earlier or later than the normal heart cells.

Taking a closer look, the scientists discovered that TBX5, NKX2.5 and GATA4 all hangout in the same areas of the genome in embryonic stem cells that are transitioning into cardiomyocytes. In fact, each individual transcription factor required the presence of the others to bind their DNA targets. If one of these factors was missing and the love triangle was broken, the remaining transcription factors became confused and bound random DNA sequences in the genome, causing a mess by turning on genes that shouldn’t be on.

First author on the study, Luis Luna-Zurita, explained the importance of maintaining this cardiac love triangle in a Gladstone Press Release:

Luis Luna-Zurita, Gladstone Institute

Luis Luna-Zurita, Gladstone Institute

“Transcription factors have to stick together, or else the other one goes and gets into trouble. Not only are these transcription factors vital for turning on certain genes, but their interaction is important to keep each other from going to the wrong place and turning on a set of genes that doesn’t belong in a heart cell.”

Crystal structure tells all

Protein crystal structure of NKX2.5 and TBX5 bound to DNA.

Protein crystal structure of NKX2.5 and TBX5 bound to DNA. (Luna-Zurita et al. 2016)

The last part of the study proved that two of these factors, NKX2.5 and TBX5, directly interact and physically touch each other when they bind their DNA targets. In collaboration with a group from the European Molecular Biology Laboratory (EMBL) in Germany, they developed protein crystal structures to model the molecular structure of these transcription factors when they bind DNA.

Co-author and EMBL scientist Christoph Muller explained his findings:

“The crystal structure critically shows the interaction between two of the transcription factors and how they influence one another’s binding to a specific stretch of DNA. Our detailed structural analysis revealed a direct physical connection between TBX5 and NKX2-5 and demonstrated that DNA plays an active role in mediating the interaction between the two proteins.”

Big picture

While this study falls in the discovery research category, its findings increase our understanding of the steps required to make a healthy heart and sheds light on what goes wrong in patients or newborns with heart disease.

Senior author on the paper and Gladstone Professor Benoit Bruneau explained the biomedical applications of their study for treating human disease:

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Benoit Bruneau, Gladstone Institute

“Gene mutations that cause congenital heart disease lower the levels of these transcription factors by half, and we’ve shown that the dosage of these factors determines which genes are turned on or off in a cell. Other genetic variants that cause heart defects like arrhythmias also affect the function of these factors. Therefore, the better we understand these transcription factors, the closer we’ll come to a treatment for heart disease. Our colleagues at Gladstone are using this knowledge to search for small molecules that can affect gene regulation and reverse some of the problems caused by the loss of these transcription factors.”

 

I think it’s worth mentioning that these studies were done using mouse embryos and mouse embryonic stem cells. Future work should be done to determine whether this cardiac love triangle and the same transcription factor interactions exist in human heart cells.

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Regenerating damaged muscle after a heart attack

/ Kevin McCormack / Leave a comment

Cardio cells image

Images of clusters of heart muscle cells (in red and green) derived from human embryonic stem cells 40 days after transplantation. Courtesy UCLA

Every year more than 735,000 Americans have a heart attack. Many of those who survive often have lasting damage to their heart muscle and are at increased risk for future attacks and heart failure. Now CIRM-funded researchers at UCLA have identified a way that could help regenerate heart muscle after a heart attack, potentially not only saving lives but also increasing the quality of life.

The researchers used human embryonic stem cells to create a kind of cell, called a cardiac mesoderm cell, which has the ability to turn into cardiomyocytes, fibroblasts, smooth muscle, and endothelial cells. All these types of cells play an important role in helping repair a damaged heart.

As those embryonic cells were in the process of changing into cardiac mesoderms, the team was able to identify two key markers on the cell surface. The markers, called CD13 and ROR2 – which makes them sound like extras in the latest Star Wars movie – pinpointed the cells that were likely to be the most efficient at changing into the kind of cells needed to repair damaged heart tissue.

The researchers then transplanted those cells into an animal model and found that not only did many of the cells survive but they also produced the cells needed to regenerate heart muscle and vessels.

Big step forward

The research was published in the journal Stem Cell Reports. Dr. Reza Ardehali, the senior author of the CIRM-funded study, says this is a big step forward in the use of embryonic stem cells to help treat heart attacks:

“In a major heart attack, a person loses an estimated 1 billion heart cells, which results in permanent scar tissue in the heart muscle. Our findings seek to unlock some of the mysteries of heart regeneration in order to move the possibility of cardiovascular cell therapies forward. We have now found a way to identify the right type of stem cells that create heart cells that successfully engraft when transplanted and generate muscle tissue in the heart, which means we’re one step closer to developing cell-based therapies for people living with heart disease.”

More good news

But wait, as they say in cheesy TV infomercials, there’s more. Ardehali and his team not only found the markers to help them identify the right kinds of cell to use in regenerating damaged heart muscle, they also found a way to track the transplanted cells so they could make sure they were going where they wanted them to, and doing what they needed them to.

In a study published in Stem Cells Translational Medicine,  Ardehali and his team used special particles that can be tracked using MRI. They used those particles to label the cardiac mesoderm cells. Once transplanted into the animal model the team was able to follow the cells for up to 40 days.

Ardehali says knowing how to identify the best cells to repair a damaged heart, and then being able to track them over a long period, gives us valuable tools to use as we work to develop better, more effective treatments for people who have had a heart attack.

CIRM is already funding a Phase 2 clinical trial, run by a company called Capricor, using stem cells to treat heart attack patients.