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Learning How Hearts Break

02/13/2013
Andrew S. Wiecek

Although researchers have learned how to direct differentiation in stem cells, the epigenetic regulation of cell lineage specification remains a mystery. But now, combining advances in stem cells and next-generation sequencing, developmental biologists are beginning to uncover some answers. Andrew S. Wiecek reports. 


At the two-week checkup for his newborn daughter, Benoit Bruneau noticed that the pediatrician was spending a lot of time listening to her heart. Just moments before, Bruneau and his wife were making small talk with one another while the doctor checked the baby’s weight, height, and head circumference. But when he began carefully listening to her heart, everything became silent. Something was wrong.

Cultured chicken embryonic cardiomyocyte transfected with GFP control vector in the nucleua, stained with alpha-actinin at the Z-lines in red and Tropomodulin1 (at the pointed ends of the actin filaments) in blue. Source: Caroline McKeown




Benoit Bruneau is the associate director at the Gladstone Institute of Cardiovascular Disease in San Francisco. His lab exclusively studies heart development, specifically looking at how cells differentiate to form the different parts of the organ during early development. Source: Twitter

Charles Murry, director of the Center for Cardiovascular Biology and co-director of the Institute for Stem Cell and Regenerative Medicine at the University of Washington, has been studying heart development since the 1980s. Source: Matthew Toles, DailyUW.com

After a quick phone call to the cardiology department at a local hospital, Bruneau and his wife took their daughter to have her heart imaged with an echocardiograph. As a result, the doctors found a medium-sized hole in the ventricular septum, the wall that divides the left and right ventricles of the heart. After learning of the news, his wife was distressed. She turned to him and asked, “Why does this happen?”

Bruneau is the associate director at the Gladstone Institute of Cardiovascular Disease in San Francisco. His lab exclusively studies heart development, specifically looking at how cells differentiate to form the different parts of the organ during early development. Awards that he has won include the Heart and Stroke Foundation of Canada New Investigator Award, Ontario’s Premier Research Excellence Award, and the Lawrence J. and Florence A. DeGeorge Charitable Trust/American Heart Association Established Investigator Award.

If anyone knew the answer to his wife’s question, it would have been Bruneau. But all of this was of little help. “I actually didn’t have an answer for her. We don’t know,” says Bruneau. “So that certainly gave me, not that I needed it, but extra motivation to understand this problem.”

Since then, aided by advances in next-generation sequencing, stem cell techniques, and bioinformatics, he has been trying to figure out what goes wrong during heart development in patients with congential heart defects and what he can do about it.

Hearts in Petri Dishes

Bruneau’s family history includes several instances of premature heart attacks, so that’s why he first became interested in matters of the heart. But somewhere along the line, he realized that if researchers could understand how the body generates myocardial cells, they could potentially use that knowledge to heal cardiac muscles after injury. So he began studying heart development and congenital heart disease.

Studying how cells differentiate during development has been a rather challenging proposition until recently. For one thing, obtaining sufficient quantities of mesodermal cells or cardiac progenitors has been difficult. Previously, researchers could extract these cells from mouse embryos, but the approach would never provide the purity or quantities of cells necessary for certain studies, such as DNA sequencing analysis. It would be possible to genetically label the cells and then sort them with flow cytometry, but this would require thousands of mouse embryos. And that’s not very feasible for most labs.

Recent advances in stem cell technology, however, have provided an alternative solution with techniques that direct the differentiation of embryonic stem cells. “We can now direct the cells towards their appropriate fate, in sufficient quantities, basically generating millions and millions of cells in a dish. It’s very efficient, and it recapitulates very well, at least as far as we know, what is happening in the embryo,” says Bruneau.

Other cardiac researchers have also realized the potential of stem cell technology to understand heart development. Charles Murry, director of the Center for Cardiovascular Biology and co-director of the Institute for Stem Cell and Regenerative Medicine at the University of Washington, has been pursuing this concept since the 1980s, back before the stem cell techniques that could make regenerative medicine possible were developed. Now, Murry’s lab has refined protocols to control the differentiation of cells to get a reasonably pure population of cardiovascular cells.

“Initially, you feel quite cheerful about it, that you’ve really accomplished something. But how much more do you really know about what differentiation is?” says Murry. “You still don’t know how this one cell can become all these different kinds of cells. And then you’re not feeling so smart anymore, and you go back to work to figure out how to peel this onion down several layers further.”

Unlike Murry’s lab, Bruneau’s group had little experience with directing stem cell differentiation. So Bruneau sent a postdoc to Gordon Keller’s lab at the University Health Network in Toronto. Keller’s research focuses primarily on lineage-specific differentiation of embryonic stem cells. When Bruneau’s postdoc returned with his schooling in stem cell differentiation, Bruneau’s team finally had the the quantity of cells needed for sequencing in place.

One Size Does Not Fit All

When you are studying development, what you are looking at is not so much the genes but rather how they are regulated through the process. One important aspect of analyzing developmental gene regulation is looking at is how epigenetic marks change at specific developmental stages of the cell. And with the technique of chromatin immunoprecipitation sequencing, or ChIP-seq for short, high-throughput sequencing can be used to study how proteins interact with DNA to regulate their transcription.

Basically, the process involves isolating and sequencing sections of DNA that are bound to proteins to understand how those proteins regulate genes. “This was the kind of experiment that is frankly impossible without high-throughput sequencing,” says Bruneau. “Sequencing allows both a genome-wide and therefore unbiased and quantitative view of occupancy of certain epigenetic modifications.”

But Bruneau’s lab had little experience with ChIP-seq. So he contacted Laurie Boyer from the Massachusetts Institute of Technology (MIT), whose lab uses genomic tools, including ChIP-seq, to study cell differentiation during development. Bruneau and Boyer are both part of the Bench to Bassinet program funded by National Heart, Lung, and Blood Institute (NHLBI). The program is part of the institute’s effort to translate basic research into treatments for congenital heart disease more quickly.

With Boyer on board, Bruneau’s lab used the stem cell-directed differentiation system to generate cells that represented four different stages during heart development. Then they packaged and shipped those cells to Boyer’s lab for ChIP-sequencing. Finally, both shared the analysis workload for the ChIP-seq data.

Likewise, when Murry was ready to look at the epigenetics of his stem cells, he turned to his Univeristy of Washington colleague John Stamatoyannopoulos who was working on the National Human Genome Research Institute’s Encyclopedia of DNA Elements project, or ENCODE for short, which looks at functional elements of the genome, gene expression, and chromatin dynamics.

But even with an expert in every aspect of these experiments, protocols for differentiation, immunoprecipitation, and sequencing still had to be optimized for these specific experiments. “There’s not a one-size-fits all for all these things, so there was a lot of optimization that had to be done. Antibodies are finicky and those sorts of things,” says Murry.

After the ChIP sequencing, the data analysis was not a walk in the park either. Looking at how chromatin marks change through differentiation seems like a simple concept, but that type of analysis has never been performed before. “It’s like getting a big block of marble; you can sculpt a variety of different stories out of it depending on the angle that you interrogate it. So we started looking for patterns,” says Murry.

And there was so much data, the biggest problem was just where to start. Both teams, however, decided to start with how chromatin is regulated in genes previously associated with heart development, such as those that encoded for the MEIS and GATA transcription factors.

Balancing Act

In back-to-back papers published in Cell in September 2012 (1-2), Bruneau and Murry reported the patterns in chromatin dynamics that emerged from their data. What both teams found was that the genes associated with cardiac-specific development and those associated with other non-specific developmental features were regulated differently. It’s almost like the genes that go on to perform a function such as contraction later in the heart are primed for later activation during early development. “We don't completely understand what that means, but it’s currently something that we are actively pursuing,” says Bruneau.

Another thing that Bruneau’s team pointed out was that activation isn’t always black and white. Previously, epigenetic researchers have found that certain genes in stem cells have both active and repressive marks as if they were pressing both the gas pedal and brake pedal at the same time. These genes have their engines racing, but another mark is holding them back. Eventually, this conflict is resolved by the gene either being completely activated or completely repressed, or so scientists believed. But Bruneau’s data shows that whether the gene is expressed or not might not be dependant on the elimination of one mark or the other but rather on the balance between the two marks.

In addition, both teams sought to identify regulators and enhancers of heart development. Overall, Murry believes that his system can identify new genes not only involved in heart development but other types of tissues as well, such as neural development and mesoderm-derived tissues.

Since the publication of these papers, Murry’s lab has been busy identifying additional factors that have not previously been associated with heat development. According to Murry, they actually have discovered a major gene that regulates heart development by controlling the Wnt signaling pathway. “It came screaming at us based on this algorithm that we developed. It was like ‘Pick me! Pick me!’ So we did, says Murry.

Vague Answers

So far, everything has only been laying the foundation for what’s next. Bruneau's group is getting ready to look at how the dynamics of epigenetic marks when development isn’t going quite right. So they’ll use stem cells that have certain cardiac transcription factors knocked out to mimic cardiac development gone awry. In addition, he plans to move into another dimension to study the 3D organization of chromatin during development to see if the spatial dynamics of enhancers and promoters can give further insights into development.

And of course, in the end, the true goal is to use that knowledge to treat patients with heart defects and disease. To further this effort, Bruneau is working with his Gladstone colleague Deepak Srivastava whose lab focuses on cellular reprogramming. In a paper published in Nature in April 2012, Srivastava and colleagues reported that they transformed cardiac fibroblasts into beating cardiomyocyte cells in a mouse model (3). This reprogramming, however, isn’t very efficient or precise yet.

Likewise, Murry’s also interested in applying what he’s learned to reprogramming cardiac cells. “I think the kind of work that we’ve done gives us a much better idea of how nature makes cardiac muscle in the first place and gives us a whole new set of candidate factors to do something like cardiac reprogramming. Once you have the fundamental knowledge, you have a whole bunch of new elements in your toolkit,” says Murry.

Every step that Bruneau and Murry take brings them closer to finding answers for patients with heart disease and for parents of children who are born with heart defects. As for Bruneau’s own daughter, the hole between her two ventricles spontaneously closed by itself. How? During the first few months of life, the heart does continue to growth. Eventually, our cardiac cells lose these capabilities. “But that’s a very vague answer,” says Bruneau. “We don’t really fully understand what happens and why in some cases it closes up and in other cases it doesn’t.”

References

1. Wamstad, J. A., J. M. Alexander, R. M. Truty, A. Shrikumar, F. Li, K. E. Eilertson, H. Ding, J. N. Wylie, A. R. Pico, J. A. Capra, G. Erwin, S. J. Kattman, G. M. Keller, D. Srivastava, S. S. Levine, K. S. Pollard, A. K. Holloway, L. A. Boyer, and B. G. Bruneau. 2012. Dynamic and coordinated epigenetic regulation of developmental transitions in the cardiac lineage. Cell 151(1):206-220.

2. Paige, S. L., S. Thomas, C. L. Stoick-Cooper, H. Wang, L. Maves, R. Sandstrom, L. Pabon, H. Reinecke, G. Pratt, G. Keller, R. T. Moon, J. Stamatoyannopoulos, and C. E. Murry. 2012. A temporal chromatin signature in human embryonic stem cells identifies regulators of cardiac development. Cell 151(1):221-232.

3. Qian, L., Y. Huang, C. I. Spencer, A. Foley, V. Vedantham, L. Liu, S. J. Conway, J.-d. Fu, and D. Srivastava. 2012. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes.Nature 485(7400):593-598.