Stem Cell Roundup: watching brain cells in real time, building better heart cells, and the plot thickens on the adult neurogenesis debate

Here are the stem cell stories that caught our eye this week.

Watching brain cells in real time

This illustration depicts a new method that enables scientists to see an astrocyte (green) physically interacting with a neuronal synapse (red) in real time, and producing an optical signal (yellow). (Khakh Lab, UCLA Health)

Our stem cell photo of the week is brought to you by the Khakh lab at UCLA Health. The lab developed a new method that allows scientists to watch brain cells interact in real time. Using a technique called fluorescence resonance energy-transfer (FRET) microscopy, the team can visualize how astrocytes (key support cells in our central nervous system) and brain cells called neurons form connections in the mouse brain and how these connections are affected by diseases like Alzheimer’s and ALS.

Baljit Khakh, the study’s first author, explained the importance of their findings in a news release:

“This new tool makes possible experiments that we have been wanting to perform for many years. For example, we can now observe how brain damage alters the way that astrocytes interact with neurons and develop strategies to address these changes.”

The study was published this week in the journal Neuron.


Turn up the power: How to build a better heart cell (Todd Dubnicoff)

For years now, researchers have had the know-how to reprogram a donor’s skin cells into induced pluripotent stem cells (iPSCs) and then specialize them into heart muscle cells called cardiomyocytes. The intervening years have focused on optimizing this method to accurately model the biology of the adult human heart as a means to test drug toxicity and ultimately develop therapies for heart disease. Reporting this week in Nature, scientists at Columbia University report an important step toward those goals.

The muscle contractions of a beating heart occur through natural electrical impulses generated by pacemaker cells. In the case of lab-grown cardiomyocytes, introducing mechanical and electrical stimulation is required to reliably generate these cells. In the current study, the research team showed that the timing and amount of stimulation is a critical aspect to the procedure.

The iPS-derived cardiomyocytes have formed heart tissue that closely mimics human heart functionality at over four weeks of maturation. Credit: Gordana Vunjak-Novakovic/Columbia University.

The team tested three scenarios on iPSC-derived cardiomyocytes (iPSC-CMs): no electrical stimulation for 3 weeks, constant stimulation for 3 weeks, and finally, two weeks of increasingly higher stimulation followed by a week of constant stimulation. This third setup mimics the changes that occur in a baby’s heart just before and just after birth.

These scenarios were tested in 12 day-old and 28 day-old iPSC-CMs. The results show that only the 12 day-old cells subjected to the increasing amounts of stimulation gave rise to fully mature heart muscle cells. On top of that, it only took four weeks to make those cells. Seila Selimovic, Ph.D., an expert at the National Institutes of Health who was not involved in the study, explained the importance of these findings in a press release:

“The resulting engineered tissue is truly unprecedented in its similarity to functioning human tissue. The ability to develop mature cardiac tissue in such a short time is an important step in moving us closer to having reliable human tissue models for drug testing.”

Read more at: https://phys.org/news/2018-04-early-bioengineered-human-heart-cells.html#jCp


Yes we do, no we don’t. More confusion over growing new brain cells as we grow older (Kevin McCormack)

First we didn’t, then we did, then we didn’t again, now we do again. Or maybe we do again.

The debate over whether we are able to continue making new neurons as we get older took another twist this week. Scientists at Columbia University said their research shows we do make new neurons in our brain, even as we age.

This image shows what scientists say is a new neuron in the brain of an older human. A new study suggests that humans continue to make new neurons throughout their lives. (Columbia University Irving Medical Center)

In the study, published in the journal Cell Stem Cell, the researchers examined the brains of 28 deceased donors aged 14 to 79. They found similar numbers of precursor and immature neurons in all the brains, suggesting we continue to develop new brain cells as we age.

This contrasts with a UCSF study published just last month which came to the opposite conclusion, that there was no evidence we make new brain cells as we age.

In an interview in the LA Times, Dr. Maura Boldrini, the lead author on the new study, says they looked at a whole section of the brain rather than the thin tissues slices the UCSF team used:

“In science, the absence of evidence is not evidence of absence. If you can’t find something it doesn’t mean that it is not there 100%.”

Well, that resolves that debate. At least until the next study.

Gladstone scientists tackle heart failure by repairing the heart from within

Modern medicine often involves the development of a drug or treatment outside the body, which is then given to a patient to fix, improve or even prevent their condition. But what if you could regenerate or heal the body using the cells and tissue already inside a patient?

Scientists at the Gladstone Institutes are pursuing such a strategy for heart disease. In a CIRM-funded study published today in the journal Cell, the team identified four genes that can stimulate adult heart muscle cells, called cardiomyocytes, to divide and proliferate within the hearts of living mice. This discovery could be further developed as a strategy to repair cardiac tissue damage caused by heart disease and heart attacks.

Regenerating the Heart

Heart disease is the leading cause of death in the US and affects over 24 million people around the world. When patients experience a heart attack, blood flow is restricted to the heart, and parts of the heart muscle are damaged or die due to the lack of oxygen. The heart is unable to regenerate new healthy heart muscle, and instead, cardiac fibroblasts generate fibrous scar tissue to heal the injury. This scar tissue impairs the heart’s ability to pump blood, causing it to work harder and putting patients at risk for future heart failure.

Deepak Srivastava, President of the Gladstone Institutes and a senior investigator there, has dedicated his life’s research to finding new ways to regenerate heart tissue. Previously, his team developed methods to reprogram mouse and human cardiac fibroblasts into beating cardiomyocytes in hopes of one day restoring heart function in patients. The team is advancing this research with the help of a CIRM Discovery Stage research grant, which will aid them in developing a gene therapy product that delivers reprogramming factors into scar tissue cells to regenerate new heart muscle.

In this new study, Srivastava took a slightly different approach and attempted to coax cardiomyocytes, rather than cardiac fibroblasts, to divide and regenerate the heart. During development, fetal cardiomyocytes rapidly divide to create heart tissue. This regenerative ability is lost in adult cardiomyocytes, which are unable to divide because they’ve already exited the cell cycle (a series of phases that a cell goes through that ultimately results in its division).

Deepak Srivastava (left) and first author Tamer Mohamed (right). Photo credits: Diana Rothery.

Unlocking proliferative potential

Srivastava had a hunch that genes specifically involved in the cell division could be used to jump-start an adult cardiomyocyte’s re-entry into the cell cycle. After some research, they identified four genes (referred to as 4F) involved in controlling cell division. When these genes were turned on in adult cardiomyocytes, the cells started to divide and create new heart tissue.

This 4F strategy worked in mouse and rat cardiomyocytes and also was successful in stimulating cell division in 15%-20% of human cardiomyocytes. When they injected 4F into the hearts of mice that had suffered heart attacks, they observed an improvement in their heart function after three months and a reduction in the size of the scar tissue compared to mice that did not receive the injection.

The team was able to further refine their method by replacing two of the four genes with chemical inhibitors that had similar functions. Throughout the process, the team did not observe the development of heart tumors caused by the 4F treatment. They attributed this fact to the short-term expression of 4F in the cardiomyocytes. However, Srivastava expressed caution towards using this method in a Gladstone news release:

“In human organs, the delivery of genes would have to be controlled carefully, since excessive or unwanted cell division could cause tumors.”

First stop heart, next stop …

This study suggests that it’s possible to regenerate our tissues and organs from within by triggering adult cells to re-enter the cell cycle. While more research is needed to ensure this method is safe and worthy of clinical development, it could lead to a regenerative treatment strategy for heart failure.

Srivastava will continue to unravel the secrets to the proliferative potential of cardiomyocytes but predicts that other labs will pursue similar methods to test the regenerative potential of adult cells in other tissues and organs.

“Heart cells were particularly challenging because when they exit the cell cycle after birth, their state is really locked down—which might explain why we don’t get heart tumors. Now that we know our method is successful with this difficult cell type, we think it could be used to unlock other cells’ potential to divide, including nerve cells, pancreatic cells, hair cells in the ear, and retinal cells.”


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Stem Cell Roundup: Improving muscle function in muscular dystrophy; Building a better brain; Boosting efficiency in making iPSC’s

Here are the stem cell stories that caught our eye this week.

Photos of the week

TGIF! We’re so excited that the weekend is here that we are sharing not one but TWO amazing stem cell photos of the week.

RMI IntestinalChip

Image caption: Cells of a human intestinal lining, after being placed in an Intestine-Chip, form intestinal folds as they do in the human body. (Photo credit: Cedars-Sinai Board of Governors Regenerative Medicine Institute)

Photo #1 is borrowed from a blog we wrote earlier this week about a new stem cell-based path to personalized medicine. Scientists at Cedars-Sinai are collaborating with a company called Emulate to create intestines-on-a-chip using human stem cells. Their goal is to create 3D-organoids that represent the human gut, grow them on chips, and use these gut-chips to screen for precision medicines that could help patients with intestinal diseases. You can read more about this gut-tastic research here.

Young mouse heart 800x533

Image caption: UCLA scientists used four different fluorescent-colored proteins to determine the origin of cardiomyocytes in mice. (Image credit: UCLA Broad Stem Cell Research Center/Nature Communications)

Photo #2 is another beautiful fluorescent image, this time of a cross-section of a mouse heart. CIRM-funded scientists from UCLA Broad Stem Cell Research Center are tracking the fate of stem cells in the developing mouse heart in hopes of finding new insights that could lead to stem cell-based therapies for heart attack victims. Their research was published this week in the journal Nature Communications and you can read more about it in a UCLA news release.

Stem cell injection improves muscle function in muscular dystrophy mice

Another study by CIRM-funded Cedars-Sinai scientists came out this week in Stem Cell Reports. They discovered that they could improve muscle function in mice with muscular dystrophy by injecting cardiac progenitor cells into their hearts. The injected cells not only improved heart function in these mice, but also improved muscle function throughout their bodies. The effects were due to the release of microscopic vesicles called exosomes by the injected cells. These cells are currently being used in a CIRM-funded clinical trial by Capricor therapeutics for patients with Duchenne muscular dystrophy.

How to build a better brain (blob)

For years stem cell researchers have been looking for ways to create “mini brains”, to better understand how our own brains work and develop new ways to repair damage. So far, the best they have done is to create blobs, clusters of cells that resemble some parts of the brain. But now researchers at the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA have come up with a new method they think can advance the field.

Their approach is explained in a fascinating article in the journal Science News, where lead researcher Bennet Novitch says finding the right method is like being a chef:

“It’s like making a cake: You have many different ways in which you can do it. There are all sorts of little tricks that people have come up with to overcome some of the common challenges.”

Brain cake. Yum.

A more efficient way to make iPS cells

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Shinya Yamanaka. (Image source: Ko Sasaki, New York Times)

In 2006 Shinya Yamanaka discovered a way to take ordinary adult cells and reprogram them into embryonic-like stem cells that have the ability to turn into any other cell in the body. He called these cells induced pluripotent stem cells or iPSC’s. Since then researchers have been using these iPSC’s to try and develop new treatments for deadly diseases.

There’s been a big problem, however. Making these cells is really tricky and current methods are really inefficient. Out of a batch of, say, 1,000 cells sometimes only one or two are turned into iPSCs. Obviously, this slows down the pace of research.

Now researchers in Colorado have found a way they say dramatically improves on that. The team says it has to do with controlling the precise levels of reprogramming factors and microRNA and…. Well, you can read how they did it in a news release on Eurekalert.

 

 

 

Making beating heart cells from stem cells just got easier

Here’s a heartwarming story for the holidays. Scientists from the Salk Institute in La Jolla, California have figured out a simple, easy way to make beating heart cells from human stem cells that will aid research and therapy development for heart disease. Their study, which received funding support from CIRM, was published last week in the journal Genes & Development.

The Salk team discovered that making beating heart tissue from human stem cells is as simple as turning off a single gene called YAP. You might be wondering how the team settled on this gene and no, it doesn’t involve pulling a random gene name out of a hat.

In previous studies, the researchers found that two cell signaling pathways, Wnt and Activin, are crucial for the development of embryonic stem cells into specialized cells like cardiomyocytes (beating heart cells). This research led to the discovery of a third pathway, controlled by YAP, which sets up a road block for cell specialization and keeps stem cells in their undifferentiated state.

Only hESCs without YAP (right panel) make heart cells (green) in one step. Blue dye marks cell nuclei. (Salk Institute)

The team deleted YAP from these stem cells using CRISPR gene editing technology, and then treated the stem cells to the Activin signaling molecule. Without YAP, exposure to Activin prompted the stem cells to develop immediately into beating cardiomyocytes that you can see beating away in the Salk video below.

Dr. Kathy Jones, Salk professor and senior author on the study, explained why this discovery is important to the field in a news release:

“This discovery is really exciting because it means we can potentially create a reliable protocol for taking normal cells and moving them very efficiently from stem cells to heart cells. Researchers and commercial companies want to easily generate cardiomyocytes to study their capacity for repair in heart attacks and disease—this brings us one step closer to being able to do that.”

First author, Conchi Estarás, emphasized how their new method for making cardiomyocytes is attractive not only for its simplicity, but also for its cost-effectiveness in enabling large-scale manufacturing of these cells for treatment.

“Instead of requiring two steps to achieve specialization, removing YAP cut it to just one step. That would mean a huge savings for industry in terms of reagent materials and expense.”

Looking ahead, Jones and her team do not plan on deleting the YAP gene from stem cells because of the potential side effects cause by the loss of YAP’s other cellular functions. Instead, they will be using commercially available molecules that can temporarily inhibit the function of YAP in hopes that this less permanent action will still readily produce beating heart cells from stem cells.

Kathy Jones and Conchi Estarás. (Image courtesy of Salk Institute)

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

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

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.

55e35-20110130_cardiomyocytes

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

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

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:

conklin-profile.jpg

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

 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:

DSC_0281_2

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

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.