Stem cell stories that caught our eye: heart muscle-on-a-chip, your own private microliver, the bloody holy grail and selfish sperm

Here are some stem cell stories that caught our eye this past week. Some are groundbreaking science, others are of personal interest to us, and still others are just fun.

Two hearts beat as one, or not
Sorry for the pre-Valentine’s Day buzzkill but stem cell research published this past week points to a very unromantic discovery: two hearts do not beat as one. The study, out of Rockefeller University, and published in the Journal of Cell Biology, sought to understand the limited success of clinical trials in which stem cell-derived heart muscle cells, or cardiomyocytes, are transplanted into the heart to help repair tissue scarred by disease or a heart attack.

If you’re a regular at The Stem Cellar, you’ll recall that just last Friday we summarized published experiments that suggest the cardiomyocytes used in successful trials do not grow new tissue themselves but instead heal the heart indirectly by releasing proteins that stimulate repair.

The research team behind this week’s study instead reasoned that the transplanted cardiomyocytes do indeed integrate into the heart tissue, but they fail to contract properly with the undamaged heart cells. So, the thinking goes, the transplanted cells do nothing to restore the heart’s ability to beat at full strength.

Watch video here: http://medicalxpress.com/news/2016-02-muscles-on-a-chip-insight-cardiac-stem.html

A two-cell “microtissue” contains a mouse embryonic stem cell-derived cardiomyocyte and a mouse neonatal cardiomyocyte. The lower panel shows the traction forces generated as the two cells contract; the stronger, neonatal cardiomyocyte produces more force than the weaker, stem cell-derived cardiomyocyte. Credit: Aratyn-Schause, Y. et al. J Cell Biol. 2016 Watch video here: http://medicalxpress.com/news/2016-02-muscles-on-a-chip-insight-cardiac-stem.html

 

To test this hypothesis, the researchers devised a two-cell micro-tissue made up of a single mouse cardiomyocyte and a single cardiomyocyte derived from either mouse embryonic stem cells or induced pluripotent stem cells (iPS). This “muscle-on-a-chip” showed that the two cells are able to physically connect up and even beat in sync with each other. But, the embryonic and iPS-derived cardiomyocytes beat less strongly than the native cell. Based on computer simulations, this imbalance made the micro-tissue beat less efficiently. A university press release picked up by Newswise includes a short yet fascinating video of the differing strengths of the beating heart cells (click on image above).

With this micro-tissue in hand, the team aims to find a way to fix this imbalance, which hopefully would make cell therapies for heart disease more potent.

Your Own Private Micro-liver
Enough about micro-hearts, let’s talk micro-livers.

In a report published on Monday in PNAS, a multidisciplinary UCSD team of engineers and biomedical researchers described the creation of a bioprinted 3D liver model made from human iPS-derived liver cells, or hepatocytes. The hepatocytes are imprinted on a surface in hexagonal shapes, the kind seen in the complex microarchitecture of the human liver. These structures were also seeded with two other cell types: endothelial cells, which form blood vessels, and fat cells, which support the health of hepatocytes. Including these relevant cell types in the “micro-liver” design resulted in a 3D cell culture that not only mimics structures but also replicates functions found in a natural liver.

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The 3-D-printed parts of the biomimetic liver tissue include: liver cells derived from human induced pluripotent stem cells (left), endothelial and mesenchymal supporing cells (center), and the resulting organized combination of multiple cell types (right). — Chen Laboratory, UC San Diego

This is a really exciting development for improving drug safety. A big concern of any new drug coming on the market is its potential liver toxicity, formally known as DILI (drug induced liver injury), the most common cause of liver failure in the U.S. Although animal studies and clinical trials carefully test for the potential of DILI, that doesn’t guarantee the drug will be safe in all individuals. And because this liver model was designed using human iPS cells – which can be derived from anyone with a simple skin biopsy – it has the potential to serve as a personalized drug screening device as well as a disease-in-a-dish model for studying inherited forms of liver disease.

As Bradley Fikes, San Diego Union Tribune’s biotechnology writer, mentions in an excellent summary of the publication, beyond drug screening and disease-in-dish modeling, this bioprinting process could also one day make it possible for researchers to reach the “holy grail” of tissue engineering: building an entire organ.

Finally! The Bloody Holy Grail
While that holy grail remains on the horizon, Stanford researchers are nearly holding the goblet in their hands. Based on a Nature report published yesterday, a team led by CIRM grantee Irv Weissman have found a long sought after cellular tag that can fish out a very specific type of hematopoietic stem cell (HSC), or blood-forming stem cell, from bone marrow.

Almost thirty years ago, Weissman identified HSCs, which have the ability to form all the cell types of the blood. Since that time, scientists have struggled with fully understanding how HSCs are maintained in the body and, in turn, how to grow them in the laboratory.

The source of this problem is due to the fact that most HSCs are so-called short term HSCs because they eventually lose their “stemness”; that is, their ability to divide indefinitely. Only a small fraction of HSCs are of the long-term variety. To really understand how the body sustains a life-long supply of HSCs, it’s necessary to have a method to pick out just the long term HSCs.

So scientists in Weissman’s lab set out to do just that. Starting with a list of 100 genes that are known to be active in the bone marrow, they looked for genes that are turned on only in long term HSCs. After a painstaking, systematic method that took two years, the team narrowed down the list to just one gene that was unique to long term HSCs.

Co-lead author James Y. Chen, a MD/PHD candidate at Stanford, described the significance of this effort in a university press release:

chen

James Y. Chen

“For nearly 30 years, people have been trying to grow HSCs outside the body and have not been able to do it — it’s arguably the ‘holy grail’ in this field. Now that we have an anchor, a way to look at long-term HSCs, we can look at the cells around them to understand and, ideally, recreate the niche.”

 

 

 

Older Dads and The Selfish Sperm

We wrap up the week with a PNAS publication that got a wide range of coverage by the likes of BBC News, Gizmodo and Cosmos in addition to the usual suspects like Health Canal. Not too surprising given the topic including selfish sperm and chopped up testicles.

Research over the past decade or so has made it increasingly clear that biological clocks not only tick for would-be moms but also dads. At first glance, it makes sense: older fathers have had more time to accumulate random DNA mutations in their spermatogonia, the stem cells that produce sperm. But studies of Apert syndrome, a rare disease causing defects in the skull, fingers and toes, has put this hypothesis in question.

Back in 2003, a research team at Oxford University found the mutation in spermatogonia that causes Apert syndrome occurs 100 to 1000 times more frequently than would be expected if it were merely due to a random mutation (the Apert syndrome is not inherited because males with the disease rarely go on to have children).

So what’s going on? To answer that question the Oxford scientists collaborated with a USC research team who (men: you may not want to read the rest of this sentence, this is your only warning) chopped up human testicles – ones that had been removed for unrelated medical reasons and donated – in order to reconstruct a three-dimensional map of where these Apert syndrome mutations were occurring. If the mutations were merely random, the affected spermatogonia would have been evenly distributed throughout the testicle. Instead, the team found clusters of cells carrying the mutation.

This results confirms a “selfish sperm” hypothesis in which the mutation provides a selective advantage to the affected sperm cells allowing them to out compete other nearby sperm cells, much like a cancer cell that multiples and gradually forms a tumor. The study serves as more sobering news to otherwise healthy older dads that they may have a higher risk of passing on harmful mutations to their offspring.

Like I said, sorry for the buzzkill. Happy Valentine’s Day weekend!

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:

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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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CIRM Scholar Jessica Gluck on using stem cells to make biological pacemakers for the heart

As part of our CIRM scholar series, we feature the research accomplishments of students and postdocs that have received CIRM funding.

Jessica Gluck, CIRM Scholar

Jessica Gluck, CIRM Scholar

I’d like to introduce you to one of our CIRM Scholars, Jessica Gluck. She’s currently a Postdoctoral Fellow at UC Davis working on human stem cell models of heart development. Jessica began her education in textiles and materials science at North Carolina State University, but that developed into a passion for biomedical engineering and stem cell research, which she pursued during her PhD at UC Los Angeles. During her graduate research, Jessica developed 3D bio-scaffolds that help human stem cells differentiate into functioning heart cells.

We asked Jessica to discuss her latest foray in the fields of stem cells and heart development.


Q: What are you currently working on in the lab?

JG: I work as a postdoc at UC Davis in the lab of Deborah Lieu. She’s working on developing pacemaking cardiomyocytes (heart cells) from human induced pluripotent stem cells (iPS cells). Pacemaking cells are the cells of the heart that are in charge of rhythm and synchronicity. Currently, we’re able to take iPS cells and get them to a cardiomyocyte state, but we want to further develop them into a pacemaking cell.

So ultimately, we’re trying to make a biological pacemaker. We can figure out how we can make a cell become the cell that tells your heart to beat, and there’s two things we can get out of that. First, if we understand how we get these beating cells, the ones that are telling the other heart cells to beat, we might be able to understand how different heart diseases progress, and we might be able to come up with a new way to prevent or treat that disease. Second, if we understand how we’re getting these pacemaking cells, we could hopefully bioengineer a biological pacemaker so you wouldn’t necessarily need an electronic pacemaker. With a biological one, a patient wouldn’t have to go back to the doctor to have their battery replaced. And they wouldn’t have to have multiple follow up surgeries throughout their life.

Q: What models are you using to study these pacemaking cells?

JG: I’m looking at my project from two different directions. On one side, we’re using a pig model, and we’re isolating cells from the sinoatrial (SA) node, which is where the pacemaking cells actually reside in your heart. And there’s really not that many of these cells. You probably have about a billion cells in your heart, but there’s maybe 100,000 of these pacemaking cells that are actually controlling the uniform beating of the heart. So we’re looking at the native SA node in the pig heart to see if it’s structurally any different than ventrical or atrial heart tissue.

Diagram of the heart depicting the Sinoatrial Node. (Image from Texas Heart Institute.

Diagram of the heart depicting the Sinoatrial Node. (Image from Texas Heart Institute)

We’ve found that the SA node is definitely different. So we’re de-cellularizing that tissue (removing the cells but not the matrix, or support structure, that keeps them in place) thinking that we could use the native matrix as a scaffold to help guide these heart cells to become the pacemaking phenotype. On the other side, we’re taking dishes with a known elasticity and we’re coating them with different proteins to see if we can tease out if there’s something that an individual protein does or a certain stiffness that actually is part of the driving force of making a pacemaking cell. We’ve gotten some pretty good preliminary results. So hopefully the next phase will be seeing how functional the cells are after they’ve been on these de-cellularized matrices.

Q: Why does your lab work with pig models?

JG: Pig hearts are pretty close to the human heart – their anatomy is pretty similar. To give you context, a pig heart is slightly larger than the size of your two hands clasped together. But the SA node, when you isolate it out, is only a couple of millimeters squared. It’s a lot smaller than we originally thought, and if we had gone with a smaller animal model, we wouldn’t be able to tangibly study or manipulate the SA node area. Because we are at UC Davis, we have a Meat Lab on campus, and we are able to get the pig hearts from them.

Q: Have you run into any road blocks with your research?

JG: For anybody that’s working with cardiomyocytes, the biggest problem is getting stem cells to become mature cardiomyocytes. Some labs have shown that you can get cells to a more mature cardiomyocyte after it’s been in culture for almost 100 days, but that’s not exactly feasible or that helpful.

We’ve been able to isolate out a small population of cells that we’re pretty sure are pacemaking cells. Over the last year, we’ve realized that a lot of the information that we thought we knew about pacemaking cells isn’t necessarily specific to pacemaking cells. Many of the biological markers that people have published in the literature are present in pacemaking cells, but we realized that they are also present in other heart cells like atrial cells, just in a lower amount. So we haven’t really been able to pick one specific biomarker that we’ve been able to say, yes this is actually a pacemaking cell. Instead, we have a small percentage of cells that we’re able to study. But we’re trying to figure out if there’s a way that we could increase our yield, or if there’s something fundamentally different about the environment that would also increase the yield of these pacemaking cells. So we’ve had a lot of trouble shooting along the way.

Q: What was your experience like as a CIRM scholar?

JG: I became a CIRM scholar in the spring of 2014. It was through the UC Davis Stem Cell Training Program. The opportunity was very helpful for me because it was in my first year as a postdoc at Davis. I earned my PhD at UCLA, so I was dealing with being on a new campus, trying to figure out whose lab I could go to to borrow random things and where to find equipment that I needed to use. So it was helpful to be around a group of other people that were also doing stem cell projects. Even though a lot of us were focused on different areas, it was still helpful to talk to other people, especially if you get somebody’s perspective that isn’t necessarily in your field. They might come up with a random idea that you haven’t thought of before.

Over the course of the year, we had a journal club, which was always interesting to see what’s going on in the field. I also went to the annual International Society for Stem Cell Research meeting in Vancouver using CIRM funding. And as part of the program, we also worked with the CIRM Bridges program between UC Davis and Cal State Sacramento. There were Bridges master’s students that were there with us. It was interesting to hear their take on everything, and they were very enthusiastic. We have had two master’s students work in our lab. I think it was very beneficial to them because they got a lot of hands on training and both have gone on to jobs in the regenerative medicine field.

Q: What is the future of stem cell research?

JG: If you’re looking at heart disease and stem cell treatments, there’s been some interesting clinical trials that have come out that have some promising results. I think that for a couple of those studies, people might have jumped the gun a little getting the treatments into the clinic. There’s still a lot that people should study in the lab before we move on to clinical trials. But I do think that we will see something in the next 20 years where stem cell research is going to have a huge therapeutic benefit. The field is just moving so quickly, and I think it will be really interesting to see what advances are made.

For our research, I’ve always been fairly realistic, and unfortunately, I don’t think we will see this biological pacemaker any time soon. But I think that the research that we produce along the way will be very beneficial to the field and our work will hopefully improve the foundation of what is known about pacemaking cells. What I think is really interesting about our lab’s work, is that we are moving into a 3D culture environment. Cells behave very differently in the body as opposed to on a plastic petri dish. So I think it’s very encouraging that we are seeing a lot more labs moving towards a more physiologically relevant model.

Q: What are your future goals?

I’ve been lucky that I’ve been able to work with very well established professors and also brand new faculty. But I’ve seen how difficult the funding climate is – it’s very daunting. So I’m really not sure what will happen next, and I’m keeping my options open.

I’ve really enjoyed working with our undergraduate and graduate students. I’ve gotten involved with outreach programs in Sacramento that promote science to young kids. It’s something that I’ve really enjoyed, and it’s very interesting telling people that I work in stem cells. Middle school kids seem to think that stem cells are magic. It’s fun to explain the very basics of stem cells and to see the light bulb moment where they understand it. I’m hoping to end up in a career that is still within the stem cell field but more towards teaching or outreach programs.

Q: What is your favorite thing about being a scientist?

JG: The thing I really like is having a puzzle that you’re trying to figure out the answer to. It’s great because every time you answer one question, that answer is going to lead you to at least three or four more new questions. I think that that’s really interesting especially trying to figure out how all the puzzle pieces fit together, and I’ve really enjoyed getting to work with people in very different fields. My parents think its funny because they said even as a little kid, I hated not knowing the answers to questions – and still do! They were completely understanding as to why I stayed in school as long as I did.

You can learn more about Jessica’s research by following her on Twitter: @JessicaGluckPhD

Timing is everything: could CRISPR gene editing push CIRM to change its rules on funding stem cell research?

CRISPR

Talk about timely. When we decided, several months ago, to hold a Standards Working Group (SWG) meeting to talk about the impact of CRISPR, a tool that is transforming the field of human gene editing, we had no idea that our meeting would fall smack in the midst of a flurry of news stories about the potential, but also the controversy, surrounding this approach.

Within a few days of our meeting lawmakers in the UK had approved the use of CRISPR for gene editing in human embryos for fertility research —a controversial first step toward what some see as a future of designer babies. And a U.S. Food and Drug Advisory report said conducting mitochondrial therapy research on human embryos is “ethically permissible”, under very limited conditions.

So it was clear from the outset that the SWG meeting was going to be touching on some fascinating and fast moving science that was loaded with ethical, social and moral questions.

Reviewing the rules

The goal of the meeting was to see if, in the light of advances with tools like CRISPR, we at CIRM needed to make any changes to our rules and regulations regarding the funding of this kind of work. We already have some strong guidelines in place to help us determine if we should fund work that involves editing human embryos, but are they strong enough?

There were some terrific speakers – including Nobel Prize winner Dr. David Baltimore; Alta Charo, a professor of Law and Bioethics at the University of Wisconsin-Madison  ; and Charis Thompson, chair of the Center for the Science, Technology, and Medicine in Society at the University of California, Berkeley – who gave some thought-provoking presentations. And there was also a truly engaged audience who offered some equally thought provoking questions.

CIRM Board member Jeff Sheehy highlighted how complex and broad ranging the issues are when he posed this question:

“Do we need to think about the rights of the embryo donor? If they have a severe inheritable disease and the embryo they donated for research has been edited, with CRISPR or other tools, to remove that potential do they have a right to know about that or even access to that technology for their own use?”

Alta Charo said this is not just a question for scientists, but something that could potentially affect everyone and so there is a real need to engage as many groups as possible in discussing it:

“How and to what extent do you involve patient advocates, members of the disability rights community and social justice community – racial or economic or geographic.  This is why we need these broader conversations, so we include all perspectives as we attempt to draw up guidelines and rules and regulations.”

It quickly became clear that the discussion was going to be even more robust than we imagined, and the issues raised were too many and too complex for us to hope to reach any conclusions or produce any recommendations in one day.

As Bernie Lo, President of the Greenwall Foundation in New York, who chaired the meeting said:

“We are not going to resolve these issues today, in fact what we have done is uncover a lot more issues and complexity.”

Time to ask tough questions

In the end it was decided that the most productive use of the day was not to limit the discussion at the workshop but to get those present to highlight the issues and questions that were most important and leave it to the SWG to then work through those and develop a series of recommendations that would eventually be presented to the CIRM Board.

The questions to be answered included but were not limited to:

1) Do we need to reconsider the language used in getting informed consent from donors in light of the ability of CRISPR and other technologies to do things that we previously couldn’t easily do?

2) Can we use CRISPR on previously donated materials/samples where general consent was given without knowing that these technologies could be available or can we only use it on biomaterials to be collected going forward?

3) Clarify whether the language we use about genetic modification should also include mitochondrial DNA as well as nuclear DNA.

4) What is the possibility that somatic or adult cell gene editing may lead to inadvertent germ line editing (altering the genomes of eggs and sperm will pass on these genetic modifications to the next generation).

5) How do we engage with patient advocates and other community groups such as the social justice and equity movements to get their input on these topics? Do we need to do more outreach and education among the public or specific groups and try to get more input from them (after all we are a taxpayer created and funded organization so we clearly have some responsibility to the wider California community and not just to researchers and patients)?

6) As CIRM already funds human embryo research should we now consider funding the use of CRISPR and other technologies that can modify the human embryo provided those embryos are not going to be implanted in a human uterus, as is the case with the recently approved research in the UK.

Stay tuned, more to come!

This was a really detailed dive into a subject that is clearly getting a lot of scientific attention around the world, and is no longer an abstract idea but is rapidly becoming a scientific reality. The next step is for a subgroup of the SWG to put together the key issues at stake here and place them in a framework for another discussion with the full SWG at some future date.

Once the SWG has reached consensus their recommendations will then go to the CIRM Board for its consideration.

We will be sure to update you on this as things progress.

How you derive embryonic stem cells matters

A scientist named James Thompson was the first to successfully culture human embryonic stem cells in 1998. He didn’t know it then, but his technique isolated a specific type of embryonic stem cell (ESC) that had a “primed pluripotent state”.

There are actually two phases of pluripotency: naïve and primed. Naïve ESCs occur a step earlier in embryonic development (during the beginning of the blastocyst stage), and the naïve state can be thought of as the ground state of pluripotency. Primed ESCs on the other hand are more mature and while they can still become every cell type in the body, they are somewhat less flexible compared to naïve ESCs. If you want to learn more about naïve and primed ESCs, you can refer to this scientific review.

Scientists have developed methods to derive both naïve and primed human ESCs in culture and are attempting to use these cells for biomedical applications. However, a recent CIRM-funded study published in Cell Stem Cell, calls into question the quality of ESCs produced using these culturing methods and could change how lab-derived stem cells are used for stem cell transplant therapies and regenerative medicine.

Primed human embryonic stem cells (purple) identified by a green stem cell surface marker. (Image courtesy of UCLA)

Primed human embryonic stem cells (purple) identified by a green stem cell surface marker. (Image courtesy of UCLA)

Culturing methods erase stem cell memory

UCLA scientists discovered that some of the culturing methods used to propagate naïve ESCs actually erase important biochemical signatures that are essential for maintaining ESCs in a naïve state and for passing down genetic information from the embryo to the developing fetus.

When they studied naïve ESCs in culture, they focused on a naturally occurring process called DNA methylation. It controls which genes are active and which are silenced by adding chemical tags to certain stretches of DNA called promoters, which are responsible for turning genes on or off. This process is critical for normal development and keeping cells functional and healthy in adults.

UCLA scientists compared the DNA methylation state of the mature human blastocyst – the early-stage embryo and where naïve ESCs come from – to the methylation state of naïve ESCs generated in culture. They found that the methylation patterns in the blastocyst six days after fertilization were the same as the patterns found in the egg that it developed from. This discovery is contrary to previous beliefs that the DNA methylation patterns in eggs are lost a few hours after fertilization.

Amander Clark, the study’s lead author and UCLA professor explained in a UCLA news release:

Amander+Clark+headshot_68295d00-2717-4d5c-99f3-f791e6b6ebcf-prv

Amandar Clark, UCLA

“We know that the six days after fertilization is a very critical time in human development, with many changes happening within that period. It’s not clear yet why the blastocyst retains methylation during this time period or what purpose it serves, but this finding opens up new areas of investigation into how methylation patterns built in the egg affect embryo quality and the birth of healthy children.”

The group also discovered cultured naïve ESCs lack these important DNA methylation patterns seen in early-stage blastocysts. Current methods to derive naïve ESCs wipe their memory leaving them in an unstable state. This is an issue for researchers because some prefer the use of naïve ESCs over primed ESCs for their studies because naïve ESCs have more potential for experimentation.

“In the past three years, naïve stem cells have been touted as potentially superior to primed cells,” Clark said. “But our data show that the naïve method for creating stem cells results in cells that have problems, including the loss of methylation from important places in DNA. Therefore, until we have a way to create more stable naïve embryonic stem cells, the embryonic stem cells created for the purposes of regenerative medicine should be in a primed state in order to create the highest-quality cells for differentiation.”

How you derive embryonic stem cells matters

Now that this culturing problem has been identified, the UCLA group plans to develop new and improved methods for generating naïve ESCs in culture such that they retain their DNA methylation patterns and are more stable.

The hope from this research is that scientists will be able to produce stem cells that more closely resemble their counterparts in the developing human embryo and will be better suited for stem cell therapies and regenerative medicine applications.


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Stem cell stories that caught our eye: affairs of the heart, better imaging of cells and pituitary glands in a dish

Here are some stem cell stories that caught our eye this past week. Some are groundbreaking science, others are of personal interest to us, and still others are just fun.

Matters of the heart not simple. The dozens of clinical trials using various types of stem cells to repair hearts after a heart attack have produced some encouraging and some discouraging results. Trials using a type of cell called a c-kit positive progenitor cells have generally produced the more positive changes in patients. But no one is completely sure why.

Get-Over-Heartbreak-Step-08The Scientist used a recent publication in Circulation Research to review the various beliefs about what these heart-derived cells do. The recent paper by University of Louisville’s Roberto Bolli confirmed what several other studies had found: the cells do not create new heart muscle themselves but do release various proteins that seem to direct natural healing. These signals, called paracrine effects, seem to last for many months even if the transplanted cells themselves don’t stick around.

The article quotes a member of a CIRM funded team using c-kit+ cells in a phase 2 clinical trial that published a paper on the paracrine effect a couple years ago:

“They’re just confirming a paradigm we and others established years ago,” said Eduardo Marban, of Cedars-Sinai hospital in Los Angeles.

The author of The Scientist piece also brings in one of the more controversial characters in the field, Piero Anversa, who while at Harvard produced a paper that suggested the c-kit+ cells could produce heart muscle, but that paper was later retracted and led Anversa to move to Switzerland from where he told the writer not all c-kit+ cells are the same. He still maintains some of the cells can produce heart muscle. This is one intrigue of the heart to be continued.

 

Hearts respond to ultrasound.  One theory on what the factors released by implanted stem cells do suggests they enlist the few cardiac stem cells we all have in our hearts. Those cells naturally try to repair the damage after a heart attack, but there just are not enough of them to be very effective. So, the paracrine factors released by donor stem cells may prod them to do a better job. Now a team in Spain suggests ultrasound treatment may do the same thing.

The researchers at the Universidad Politecnica de Madrid applied low-intensity pulsed ultrasound in mice with damaged hearts and found improved performance of the heart stem cells. Laboratory tests suggested the ultrasound treatment improved the cells mobility, in effect made them better able move to the site of damage. MedicalNews.net picked up a piece from the university on the research.

 

Give those cells a hug.  Most of you have seen the many colorful images we post of stem cells with various parts of the cells glowing in different colors. These fluorescent tags on specific proteins in the cells help scientists identify and track cellular bits of interest. They sometimes introduce the tags genetically during development of the cells, but if they want to introduce them into living tissue after the fact, they have trouble getting the often large fluorescent tags into cells in a way that maintains a living cell’s normal function.

compressingc

Proteins labeled using the new technique that compresses the cells to create pores

Now teams working at MIT and Goethe University in Frankfurt have refined a technique that squeezes living cells and creates temporary pores that lets the tags into the cells. The teams published their work in Nature Communications and a press release from MIT picked up by Phys.Org offered a quote from one of the authors, Armon Sharei, on the value of the work to the field in general:

“Basically everything that happens in your cells is mediated by proteins. You can start to learn a lot about the basic biology of how a cell works, how it divides, and what makes the cancer cell a cancer cell, as far as what mechanisms go awry and what proteins are responsible for that.”

Another mini-organ, the pituitary.  The list of miniature organs created in the lab has grown to at least a dozen with the creation of pituitary glands by a team at Japan’s RIKEN Center, where some of the other “organoids” have been made. Because the pituitary is tiny, the lab grown version comes closer to the size of the natural one, and may be ready for clinical consideration sooner.

The pituitary gland secretes several hormones that control bodily functions, and when it is out of whack, you really know it. So a replacement would be a boon for patients, who now receive hormone replacement therapy that is not fully effective.

SciCasts wrote a story on the research that provides a nice narrative of the various steps the researchers took to get to a functional mini-organ that worked to correct hormone level when implanted in mice. It ends with a quote from Takashi Tsuji the head of the appropriately named Laboratory for Organ Regeneration:

“This is an exciting step forward toward our ultimate goal, which is to be able to regrow fully functioning organs in the laboratory. We will continue to push ahead with experiments to grow other parts of the body.”

Sushi Just Got Even Better: Gel Made from Seaweed Improves the Shelf-Life of Stem Cells

The beauty of pharmaceutical drugs is their stability. Those ibuprofen pills in your medical cabinet can sit there for weeks, months, even years but still dull a sudden headache.

200mg_ibuprofen_tablets

Unlike ibuprofen pills, you can’t store stem cells in your medicine cabinet (Photo: Wikimedia Commons)

Stem cell-based therapies don’t have that luxury because, well, they’re made of living cells. Outside of the body, cells are the opposite of stable. To keep them alive and active, they need to be maintained in a precise mix of liquid nutrients and in a controlled environment of 98.6F with 5% oxygen, or frozen.

Fragile: handle with care
This tight set of conditions makes the processing, storage and transport of cell therapies to doctors and their patients tricky. Studies have shown that frozen bone marrow-derived stem cells start dying within two hours after thawing. Refrigerating them instead isn’t much better. In that case, cells could be stably held for 6-8hr. These methods don’t leave much time for carrying out the logistics of cell-based treatments and leave questions about the actual dose of intact cells in each treatment batch.

Based on a new report in Stem Cells Translational Medicine, scientists at Newcastle University in the UK have identified a method for improving the shelf-life of stem cells. This finding could go a long way toward lengthening the time window that a cell therapy dose remains intact which in turn will help lower manufacturing and treatment costs.

Sushi just got even better
The team focused on stem cells found in human adipose, or fat, tissue. These human adipose stem cells (hASCs) are a good source of mesenchymal stem cells (MSCs) which are known for their anti-inflammatory and wound healing effects, among other things. The team tested the impact on cell stability when storing them in alginate, a gel-like substance found in seaweed. They ran the test over three days at various temperatures ranging from about 40F to 75F. Here’s a 60 second video showing the alginate technique:

The tests showed that when stored in alginate between 52F and 66F, greater than 70% of the cells were recovered after three days with a max recovery of 86% at 59F. That 70% number threshold looms large because it’s the Food and Drug Administration’s (FDA) minimum acceptable recovery rate for cellular products. In comparison, none of the cell batches stored without alginate reached a 70% recovery no matter which temperature was tested.

The hASCs stored in alginate not only survived well they also functioned just as well as the cells stored without alginate. When transferred back to petri dishes, they still had the capacity to divide and their ability to be specialized into fat, bone and cartilage cells remained intact.

So what exactly is the “secret sauce” behind alginate’s protective effect? A Newcastle University press release summarized the teams’ idea of how the alginate “stem gel” works:

“They believe it may be acting like a corset, preventing the stem cell from expanding and being destroyed, a process known as lysing – which would normally occur within a day when unprotected cells are stored in their liquid state.”

Stem cell Band-Aid
And because the cells survived well even at room temperature while embedded in the alginate, the team has developed an exciting application, which Che Connon, the team’s leader and professor of tissue engineering, explained in the press release:

“This has lots of advantages and applications. For example, we have used them to make a bandage which contains human stem cells which could be applied to a wound such as an ulcer or burn to speed up the healing process.”

stemgellbandaid.jpg

Stem cells stored in alginate gel are stable at room temperature enabling a stem cell bandage for improved wound healing. Photo: Newcastle University, Mike Urwin.

The big picture
Although it may not be as glamorous as finding a cell therapy cure for a deadly disease, this alginate storage technique and others like it become critical for keeping the cell therapy supply chain streamlined, quality-controlled and cost-effective – especially as more and more stem cell-based clinical trials come on line and begin reaching approval for the general population.

A Tale of Two Stem Cell Treatments for Growing New Bones

Got Milk?

GotmilkIf you grew up during the 90’s, you most certainly will remember the famous “Got Milk?” advertising campaign to boost milk consumption. The plug was that milk was an invaluable source of calcium, a mineral that’s essential for growing strong bones. Drinking three glasses of the white stuff a day, supposedly would help deter osteoporosis, or the weakening and loss of bone with old age.

Research has proven that calcium is essential for growing and maintaining healthy bones. But milk isn’t the only source of calcium in the human diet, and a diet rich in calcium alone won’t prevent everyone from experiencing some amount of bone loss as they grow older. It also won’t help patients who suffer from bone skeletal defects grow new bone.

So whatever are we to do about bone loss and bone abnormalities? Here, we tell the “Tale of two stem cell treatments” where scientists tackle these problems using stem cell-derived therapies.

Protein Combo Boosts Bone Growth

Osteoporosis. (Image source)

Osteoporosis. (Image source)

Our first story comes from a CIRM-funded team of UCLA scientists. This team is interested in developing a better therapy to treat bone defects and osteoporosis. The current treatment for bone loss is an FDA-approved bone regenerating therapy involving the protein BMP-2 (bone morphogenetic protein-2). The problem with BMP-2 is that it can cause serious side effects when given in high doses. Two of the major ones are abnormal bone growth and also making stem cells turn into fat cells as well as bone cells.

The UCLA group attempted to improve the BMP-2 treatment by adding a second protein called NELL-1 (which they knew was good at stimulating bone growth from previous studies).  The combination of BMP-2 and NELL-1 resulted in bone growth and also prevented stem cells from making fat cells.

Upon further exploration, they found that NELL-1 acts as a signaling switch that controls whether a stem cell becomes a bone cell or a fat cell. Thus, with NELL-1 present, BMP-2 can only turn stem cells into bone cells.

Kang Ting, a lead author on the study, explained the significance of their new strategy to improve bone regeneration in a UCLA press release:

Kang Ting, UCLA

Kang Ting, UCLA

“Before this study, large bone defects in patients were difficult to treat with BMP2 or other existing products available to surgeons. The combination of NELL-1 and BMP2 resulted in improved safety and efficacy of bone regeneration in animal models — and may, one day, offer patients significantly better bone healing.”

Chia Soo, another lead author on the study, emphasized the importance of using NELL-1 in combination with BMP-2:

“In contrast to BMP2, the novel ability of NELL-1 to stimulate bone growth and repress the formation of fat may highlight new treatment approaches for osteoporosis and other therapies for bone loss.”

Stem cells that could fix deformed skulls

Our second story comes from a group at the University of Rochester. Their goal is to repair bones in the face and skull of patients suffering from congenital deformities, or damage due to injury or cancer surgery.

In a report published in Nature Communications, the scientists identified a population of skeletal stem cells that orchestrate the formation of the skull and can promote craniofacial bone repair in mice.

They identified this special population of skeletal stem cells by their expression of a protein called Axin2. Genetic mutations in the Axin2 gene can cause a birth defect called craniosynostosis. This condition causes the bone plates of a baby’s skull to fuse too early, causing skull deformities and impaired brain development.

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Axin2 stem cells shown in red and blue generated new bones cells after transplantation.

According to a news release from the University of Rochester, the group’s “latest evidence shows that stem cells central to skull formation are contained within Axin2 cell populations, comprising about 1 percent—and that the lab tests used to uncover the skeletal stem cells might also be useful to find bone diseases caused by stem cell abnormalities.”

Additionally, senior author on the study, Wei Hsu, “believes his findings contributee to an emerging field involving tissue engineering that uses stem cells and other materials to invent superior ways to replace damaged craniofacial bones in humans due to congenital disease, trauma, or cancer surgery.”

Two different studies, one common goal

Both studies have a common goal: to repair or regenerate bone to treat bone loss, damage, or deformities. I can’t help but wonder whether these different strategies could be combined in a way to that would bring more benefit to the patient than using either strategy alone.

Could we use BMP-2 and NELL-1 treatment along with Axin2 skeletal stem cells to treat craniosynostosis or repair damaged skulls? Or could we identify new stem cell populations in bone that would help patients suffering from osteoporosis?

I’m sure scientists will answer these questions sooner rather than later, and when they do, you’ll be sure to read about it on the Stem Cellar!


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If you want to accelerate stem cell therapies then create an Accelerating Center

Buckle up

Buckle up, we’re about to Accelerate

“You can’t teach fish to fly,” is one of the phrases that our CIRM President & CEO, Randy Mills, likes to throw out when asked why we needed to create new centers to help researchers move their most promising therapies out of the lab and into clinical trials.

His point is that many researchers are terrific at research but not so great at the form filling and other process-oriented skills needed to get approval from the Food and Drug Administration (FDA) for a clinical trial.

So instead of asking them to learn how to do all those things, why don’t we, CIRM, create a system that will do it for them? And that’s where we came up with the idea for the Accelerating Center (we’re also creating a Translating Center – that’s a topic for a future blog but if you can’t wait to find out the juicy details you can find them here.)

The Accelerating Center will be a clinical research organization that provides regulatory, operational and other support services to researchers and companies hoping to get their stem cell therapies into a clinical trial. The goal is to match the scientific skills of researchers with the regulatory and procedural skills of the Accelerating Center to move these projects through the review process as quickly as possible.

But it doesn’t end there. Once a project has been given the green light by the FDA, the Accelerating Center will help with actually setting up and running their clinical trial, and helping them with data management to ensure they get high quality data from the trial. Again these skills are essential to run a good clinical trial but things researchers may not have learned about when getting a PhD.

We just issued what we call an RFA (Request for Applications)  for people interested in partnering with us to help create the Accelerating Center. To kick-start the process we are awarding up to $15 million for five years to create the Center, which will be based in California.

To begin with, the Accelerating Center will focus on supporting CIRM-funded stem cell projects. But the goal is to eventually extend that support to other stem cell programs.

Now, to be honest, there’s an element of self-interest in all this. We have a goal under our new Strategic Plan of funding 50 new clinical trials over the next five years. Right now, getting a stem cell-related project approved is a slow and challenging process. We think the Accelerating Center is one tool to help us change that and give the most promising projects the support they need to get out of the lab and into people.

There’s a lot more we want to do to help speed up the approval process as well, including working with the FDA to create a new, streamlined regulatory process, one that is faster and easier to navigate. But that may take some time. So in the meantime, the Accelerating Center will help “fish” to do what they do best, swim, and we’ll take care of the flying for them.

 

 

 

CREATE-ing tools that deliver genes past the blood-brain barrier

Your brain has a natural defense that protects it from infection and harm, it’s called the blood-brain barrier (BBB). The BBB is a selectively permeable layer of tightly packed cells that separates the blood in your circulatory system from your brain. Only certain nutrients, hormones, and molecules can pass through the BBB into the brain, while harmful chemicals and infection-causing bacteria are stopped at the border.

This ultimate defense barrier has its downsides though. It’s estimated that 98% of potential drugs that could treat brain diseases cannot pass through the BBB. Only some drug compounds that are very small in size or are fat-soluble can get through. Clearly, getting drugs and therapies past the BBB is a huge conundrum that remains to be solved.

Penetrating the Impenetrable

However, a CIRM-funded study published today in Nature Biotechnology has developed a delivery tool that can bypass the BBB and deliver genes into the brain. Scientists from Caltech and Stanford University used an innocuous virus called an adeno-associated virus (AAV) to transport genetic material through the BBB into brain cells.

Viral delivery is a common method to target and deliver genes or drugs to specific tissues or cells in the body. But with the brain and its impenetrable barrier, scientists are forced to surgically inject the virus into specific areas of the brain, which limits the areas of the brain that get treatment, not to mention the very invasive and potentially damaging nature of the surgery itself. For diseases that affect multiple areas in the brain, like Huntington’s and Alzheimer’s disease, direct injection methods are not likely to be effective. Thus, a virus that can slip past the BBB and reach all parts of the brain would be an idea tool for delivering drugs and therapies.

And that’s just what this new study accomplished. Scientists developed a method for generating modified AAVs that can be injected into the circulatory system of mice, pass through the BBB, and deliver genetic material into the brain.

They devised a viral selection assay called CREATE (which stands for Cre Recombinase-based AAV Targeted Evolution). Using CREATE, they tested millions of AAVs that all had slight differences in the genetic composition of their capsid, or the protein shell of the virus that protects the viruses’ genetic material. They tested these modified viruses in mice to see which ones were able to cross the BBB and deliver genes to support cells in the brain called astrocytes. For more details on how the science of CREATE works, you can read an eloquent summary in the Caltech press release.

A Virus that Makes Your Brain Glow Green

After optimizing their viral selection assay, the scientists were able to identify one AAV in particular, AAV-PHP.B, that was exceptionally good at getting past the BBB and targeting astrocytes in the mouse brain.

Lead author on the study, Ben Deverman, explained: “By figuring out a way to get genes across the blood-brain barrier, we are able to deliver them throughout the adult brain with high efficiency.”

They used AAV-PHP.B and AAV9 (which they knew could pass the BBB and infect brain cells) to transport a gene that codes for green fluorescent protein (GFP) into the mouse brain. After injecting mice with both viruses containing GFP, they saw that both viruses were able to make most of the cells in the brain glow green, confirming that they successfully delivered the GFP gene. When they compared the potency of AAV-PHP.B to the AAV9 virus, they saw that AAV-PHP.B was 40 times more efficient in delivering genes to the brain and spinal cord.

sing a new selection method, Caltech researchers have evolved the protein shell of a harmless virus, AAV9, so that it can more efficiently cross the blood brain barrier and deliver genes, such as the green fluorescent protein (GFP), to cells throughout the central nervous system. Here, GFP expression in naturally occurring AAV9 (left) can be seen distributed sparsely throughout the brain. The modified vector, AAV-PHP.B (right), provides more efficient GFP expression. Credit: Ben Deverman and the Gradinaru laboratory/Caltech - See more at: http://www.caltech.edu/news/delivering-genes-across-blood-brain-barrier-49679#sthash.BDu7OfC8.dpuf

Newly “CREATEd” AAV-PHP.B (right) is better at delivering the GFP gene to the brain than AAV9 (left). Credit: Ben Deverman.

“What provides most of AAV-PHP.B’s benefit is its increased ability to get through the vasculature into the brain,” said Ben Deverman. “Once there, many AAVs, including AAV9 are quite good at delivering genes to neurons and glia.”

Senior author on the study, Viviana Gradinaru at Caltech, elaborated: “We could see that AAV-PHP.B was expressed throughout the adult central nervous system with high efficiency in most cell types.”

Not only that, but using a neat technique called PARS CLARITY that Gradinaru developed in her lab, which makes tissues and organs transparent, the scientists were able to see the full reach of the AAV-PHP.B virus. They saw green cells in other organs and in the peripheral nerves, thus showing that AAV-PHP.B works in other parts of the body, not just the brain.

But just because AAV-PHP.B is effective in mice doesn’t mean it works well in humans. To address this question, the authors tested AAV-PHP.B in human neurons and astrocytes derived from human induced pluripotent stem cells (iPS cells). Sergiu Pasca, a collaborator from Stanford and author on the study, told the Stem Cellar:

Sergiu Pasca

Sergiu Pasca

“We have also tested the new AAV variant (AAV-PHP.B) in a human 3D cerebral cortex model developed from human iPS cells and have shown that it transduces human neurons and astrocytes more efficiently than does AAV9 demonstrating the potential for biomedical applications.”

An easier way to deliver genes across the BBB

This study provides a new way to cross the BBB and deliver genes and potential therapies that could treat a laundry list of degenerative brain diseases.

This is only the beginning for this new technology. According to the Caltech press release, the study’s authors have future plans for the AAV-PHP.B virus:

“The researchers hope to begin testing AAV-PHP.B’s ability to deliver potentially therapeutic genes in disease models. They are also working to further evolve the virus to make even better performing variants and to produce variants that target certain cell types with more specificity.”


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