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

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.”


Related Links:

Stem cell stories that caught our eye: watching tumors grow, faster creation of stem cells, reducing spinal cord damage, mini organs

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.

Video shows tumors growing. A team at the University of Iowa used video to capture breast cancer cells recruiting normal cells to the dark side where they help tumors grow.

Led by David Soll, the team reports that cancer cells secrete a cable that can reach out and actively grab other cells. Once the cable reaches another cell, it pulls it in forming a larger tumor.

 “There’s nothing but tumorigenic cells in the bridge (between cells),” Soll said in a story in SciCasts, “and that’s the discovery. The tumorigenic cells know what they’re doing. They make tumors.”

They published their work in the American Journal of Cancer Research, and in a press release they suggested the results could provide an alternative to the theory that cancer stem cells are the engine of tumor growth.  I would guess that before too long, someone will find a way to merge the two theories into one, more cohesive story of how cancer grows.

 

3-D home creates stem cells quicker. Using a 3-D gel to grow the cells, a Swiss team reprogrammed skin cells into iPS-type stem cells in half the time that it takes in a flat petri dish. Since these induced Pluripotent Stem cells have tremendous value now in research and potentially in the future treating of patients, this major improvement in a process that has been notoriously slow and inefficient is great news.

The senior researcher Matthias Lutoff from Polytechnique Federale explained that the 3-D environment gave the cells a home closer to the environment where they would grow in someone’s body. In an article in Healthline, he described the common method used today:

 “What we currently have available is this two dimensional plastic surface that many, many stem cells really don’t like at all.”

At CIRM our goal is to get this research done as quickly as possible and to find ways to scale up any therapy so that it becomes practical to make it available to all patients who need it. Healthline quoted our CIRM scientist colleague Kevin Whittlesey on how the work would be a boon for stem cells scientists with its ability to shave months off the process of creating iPS cells.

 

Help for recent spinal cord injury.  A team at Case Western Reserve University in Cleveland used the offspring of stem cells that they are calling multi-potent adult progenitor cells (MAPCs) to modulate the immune response after spinal cord injury. They wanted to preserve some of the role of the immune system in clearing debris after an injury but prevent any overly rambunctious activity that would result in additional damage to healthy tissue and scarring.

a6353-spinalcord

They published their work in Scientific Reports and at the web portal MD the senior researcher Jerry Silver described the project as targeting a specific immune cell, the macrophage, in the early days following stroke in mice:

 

 “These were kinder, gentler macrophages. They do the job, but they pick and choose what they consume. The end result is spared tissue.”

The team injected the MAPCs into the mice one day after injury. Those cells were observed to go mostly to the spleen, which is know to be a reservoir for macrophages, and from their the MAPCs seemed to modulate the immune response.

 “There was this remarkable neuroprotection with the friendlier macrophages,” Silver explained. “The spinal cord was just bigger, healthier, with much less tissue damage.”

 

Rundown on all the mini-organs.  Regular readers of The Stem Cellar know researchers have made tremendous strides toward growing replacement organs from stem cells. You also know that with a few exceptions, like bladders and the esophagus, these are not ready for transplant into people.

Live Science web site does a fun rundown of progress with 11 different organs. They hit the more advanced esophagus and cover the early work on the reproductive tract, with items on fallopian tubes, vaginas and the penis. But most of the piece covers the early stage research that results in mini-organs, or as some have dubbed them, organoids. The author includes brain, heart, kidney, lung, stomach and liver. They also throw it the recent full ear grown on a scaffold.

Each short item comes with a photograph, mostly beautiful fluorescent microscopic images of cells forming the complex structures that become rudimentary organs.

3D printed human ear.

3D printed human ear.

Mini-stomachs.

Mini-stomachs.

This past summer we wrote about an article on work at the University of Wisconsin on the many hurdles that have to be leapt to get actual replacement organs. Progress is happening faster that most of us expected, but we still have a quite a way to go.

From Science Fiction to Science Fact: Gene Editing May Make Personalized Therapies for Blindness

Have you seen the movie Elysium? It’s a 2013 futuristic science fiction film starring one of my favorite actors Matt Damon. The plot centers on the economic, social and political disparities between two very different worlds: one, an overpopulated earth where people are poor, starving, and have little access to technology or medical care, the other, a terraformed paradise in earth’s orbit that harbors the rich, the beautiful, and advanced technologies.

Med-Bays.

Med-Bays.

The movie is entertaining (I give it 4 stars, Rotten Tomatoes says 67%), but as a scientist, one of the details that stuck out most was the Med-Bays. They’re magical, medical machines that can diagnose and cure any disease, regrow body parts, and even make people young again.

Wouldn’t it be wonderful if Med-Bays actually existed? Unfortunately, we currently lack the capabilities to bring this technology out of the realm of science fiction. However, recent efforts in the areas of personalized stem cell therapies and precision medicine are putting paths for creating potential cures for a wide range of diseases on the map.

One such study, published in Scientific Reports, is using precision medicine to help cure patients with a rare eye disease. Scientists from the University of Iowa and Columbia University Medical Center used CRISPR gene editing technology to fix induced pluripotent stem cells (iPS cells) derived from patients with an inherited form of blindness called X-linked retinitis pigmentosa (XLRP). The disease is caused by a single genetic mutation in the RPGR gene, which causes the retina of the eye to break down, leaving the patient blind or with very little vision. (For more on RP and other diseases of blindness, check out our Stem Cells in your Face video.)

CRISPR is a hot new tool that allows scientists to target and change specific sequences of DNA in the genome with higher accuracy and efficiency than other gene editing tools. In this study, researchers were concerned that it would be hard for CRISPR to correct the RPGR gene mutation because it’s located in a repetitive section of DNA that can be hard to accurately edit. After treating patient stem cells with the CRISPR modifying cocktail, the scientists found that the RPGR mutation had a 13% correction rate, which is comparable to other iPS cell based CRISPR editing studies.

Skin cells from a patient with X-linked Retinitis Pigmentosa were transformed into induced pluripotent stem cells and the blindness-causing point mutation in the RPGR gene was corrected using CRISPR/Cas9. Image by Vinit Mahajan.

Stem cells derived from a patient with X-linked Retinitis Pigmentosa. (Image by Vinit Mahajan)

The authors claim that this is the first study to successfully correct a genetic mutation in human stem cells derived from patients with degenerative retinal disease. The study is important because it indicates that XLRP patients can benefit from personalized stem cell therapy where scientists make individual patient iPS cell lines, use precision medicine to genetically correct the RPGR mutation, and then transplant healthy retinal cells derived from the corrected stem cells back into the same patients to hopefully give them back their sight.

Senior author on the study, Vinit Mahajan explained in a University of Iowa news release:

Vinit Mahajan

Vinit Mahajan

“With CRISPR gene editing of human stem cells, we can theoretically transplant healthy new cells that come from the patient after having fixed their specific gene mutation. And retinal diseases are a perfect model for stem cell therapy, because we have the advanced surgical techniques to implant cells exactly where they are needed.”

It’s important to note that this study is still in its early stages. Stephen Tsang, a co-author on the study, commented:

“There is still work to do. Before we go into patients, we want to make sure we are only changing that particular, single mutation and we are not making other alterations to the genome.”


Related Links:

Stem cell stories that caught our eye: colon cancer relapse and using age, electricity and a “mattress” to grow better hearts

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.

Stem cells yield markers for relapse in colon cancer. Some colon cancer patients do fine after surgery without any chemotherapy, but it has been hard to predict which ones. A CIRM-funded team at the University of California, San Diego, with collaborators at Stanford and Columbia Universities, found a predictor for the need for chemotherapy by looking at the patients’ cancer stem cells.

colon cancer stem cells

Patients whose colon cancer stem cells tested positive for CDX2 (brown) had a better prognosis.

Previously researchers have looked for markers in the tumors themselves for differences between those who require chemotherapy and those who don’t. Those efforts generally come up empty handed. The current team instead looked for differences in the patient’s cancer stem cells. They found that patients whose stem cells lacked one protein marker called CDX2 did poorer with surgery alone and were candidates for follow-up chemotherapy.

The team published its work in this week’s New England Journal of Medicine and it got wide pickup by online news outlets, but that coverage varied somewhat depending on which group the reporters called. Medical News Today provides the Columbia angle. Newswise distributed a press release with the San Diego voice and BlackDoctor.org used quotes from Stanford as well as the American Cancer Society. The latter lets Stanford’s Michael Clark remind readers that this was a retrospective look back at prior cancer patients and the conclusions need confirmatory studies.

 “The data is extremely strong, but you need a prospective analysis to be 100 percent sure. It should be validated in a prospective trial.”

 

Three studies aim for better heart cells. While researchers have been turning stem cells into heart muscle in lab dishes for several years, getting them to function like normal heart cells either in the dish or when transplanted into animals has been tough. Three research groups published studies this week showing different approaches to making better heart muscle.

normal heart cells

Normal heart muscle cells, courtesy Kyoto University

Age matters

 Biologists at Japan’s Kyoto University found a sweet spot in the age of new muscle cells when they were most likely to engraft and survive when transplanted in animals. They first created reprogrammed iPS-type stem cells and then matured them toward becoming heart muscle for four, eight, 20 and 30 days. The 20-day cells proved the most able to engraft in the mouse hearts and improve their function as seen by echocardiography.

The Kyoto team published its results in Scientific Reports and BiotechDaily wrote an article on the work.

Give them a jolt. 

A group of physician engineers at Columbia University found that exposing lab grown heart muscle cells to electrical stimulation that mimicked the signals the cells would receive in a fetus resulted in stronger, more synchronized heart muscle. They started by engineering the heart muscle cells to grow in three dimensions and then added the electrical signals.

 “We applied electrical stimulation to mature these cells, regulate their contractile function, and improve their ability to connect with each other. In fact, we trained the cell to adopt the beating pattern of the heart, improved the organization of important cardiac proteins, and helped the cells to become more adult-like,” said Gordana Vunjak-Novakovic, the lead author on the paper published in Nature Communications.

 NewsMedical picked up the university’s press release.

Give them a mattress. 

 A team at Vanderbilt University in Tennessee found that growing the heart muscle cells on a commonly used lab gel called Matrigel resulted in cells with a shape and contractile function that matched normal heart tissue. The Matrigel formed a cushiony substrate that one team member referred to as a “mattress” for the cells to grow on that is more like the living environment in an animal than the usual lab dish.

ScienceDaily ran the university’s press release about the study published in Circulation Research. In the release, the team speculated that the matrigel worked through a combination of the flexibility of the gel and unknown growth factors released by the gel itself.

With heart disease still a leading cause of death, learning how to make better repair tissue could lead to major improvements in quality and length of lives. Of the 600-plus stem cell clinical trails currently active around the world, at least 70 target heart disease, but very few are striving to provide new tissue to repair damaged heart muscle. Generally, they are using stem cells that secrete various factors that help the heart heal itself. CIRM funds one of those trials being conducted by Capricor.

Computer “Magic” Helps Scientists Morph One Cell’s Identity Into Another

Mogrify. Sounds like one of Harry Potter’s spells, doesn’t it? In reality, it’s something cooler than that. As reported on Tuesday in Nature Genetics, Mogrify is a new research tool that uses the magic of mathematics and computer programming to help stem cell scientists determine the necessary ingredients to convert one human cell type into another.

mogrifyharrypotter

It may sound like a magical spell but Mogrify is based on real science to help researchers predict what factors are needed to convert a given cell into another. Image credit: Warner Bros.

Now, make no mistake, the stem cell field already has the knowhow to manipulate the identity of cells and stem cells in order to study human disease and work toward cell therapies. Got a human embryonic stem cell? Scientists can specialize, or differentiate, that into an insulin-producing pancreatic cell or a beating heart muscle cell to name just two examples. Got a skin cell from an autistic patient? Using the induced pluripotent stem cell (iPS) technique, researchers have worked out the steps to transform that skin cell into an embryonic stem cell-like state and then differentiate it to a nerve cell – providing new insights into the disorder. This iPS technique can even be skipped altogether to directly convert a skin cell into, say, a liver cell through a technique called transdifferentiation.

But these methods require trial and error to pinpoint the right combination of genetic on/off switches to “flip” in the cells. These switches are called transcription factors, proteins that bind to DNA and activate or repress genes. The interaction between transcription factors and genes that give a cell it’s specific identity is extremely complex. To mimic these interactions in a lab dish, scientists use their expert knowledge and make educated guesses about which combinations of genes to modulate to generate certain cell types. Still, trial and error is a necessary part of the workflow which can require months and even years of work. And with about 2000 transcription factors and 400 cell types in humans, there’s an enormous number of possible combinations to potentially test.

Meet Mogrify
This is where Mogrify, a computational algorithm developed by a collaboration between scientists at the University of Bristol in the UK and Monash University in Australia, comes into the picture. Without lifting a pipette, Mogrify appears to be able to determine the most likely combination of transcription factors to transdifferentiate a given cell type into another without forcing the cell back to an embryonic stem cell state.

Mogrify was applied to FANTOM5, a dataset created by a large international effort to describe gene activity networks in all the cell types of the human body. With Mogrify and FANTOM5 in hand, the team first validated their algorithm by making predictions for transdifferentiation recipes that have already been established in scientific publications. For example, Mogrify correctly predicted that the transcription factor, MYOD1, could directly convert a skin cell to a muscle cell, one of the early examples of transdifferentiated cells described back in the 1980’s by the lab of Harold Weintraub. Altogether these “in silico” validation experiments recovered the correct published transcription factors at a rate of 84% compared to 31% and 51% for two other computer algorithms published by independent groups. And in 6 out of the 10 conversion experiments, Mogrify predicted 100% of the required transcription factors. As the team points out in their research article, had Mogrify been available to these scientists, they would have saved a lot of time:

“If Mogrify had been used in the original studies, the experiments could have been a success the first time.”

In addition to these validation tests, the team also tried out Mogrify in lab experiments without the help of previous publications. In one of the experiments they asked Mogrify to suggest transdifferentiation factors for converting adult fibroblasts, which are collagen-producing cells, into keratinocytes, the cells that make up the outer layer of our skin.  The algorithm predicted a set of five transcription factors which were then introduced into the fibroblasts in the lab. Within three weeks, most of the fibroblasts had converted into cells resembling keratinocytes – they had the appropriate protein markers on their surface and had taken on the typical shape seen in keratinocytes.

mogrify

The image shows the results of converting fibroblasts (collagen producing cells) to keratinocytes (skin cells) using the Mogrify algorithm. In the image it can be seen that the converted keratinocytes, which are stained green, have a ‘cobble-stone’ pattern while fibroblasts have a long thin morphology. Credit: Nature Genetics & Rackham et al.

Insights and Questions
I think Mogrify is a fascinating example of how machines and human brain power together can push the envelope of biological discoveries. Through laboratory research, scientists gradually build mental models of various cellular processes. These mental models are sources of thought experiments that they test in the lab. Yet, the countless interactions between genes, proteins and cells is so complex that the intuition of even the greatest scientific minds breaks down at some point. That’s where researchers can leverage the insight of tools like Mogrify.

Will Mogrify be a breakthrough game-changer in the world of stem cell science? Only time will tell as more scientists around the world put it to use. And thanks to the team, one can start using it right now because it’s available to anyone online. Just select your starting and finishing cell types from a pull down menu to begin.

mogrify_screenshot2

Screenshot from Mogrify.net. Just select your desired starting and finishing cell types and Mogrify recommends which transcription factors to use for your cell conversion. 

Will Mogrify completely eliminate the need to do some trial and error? Not likely, as the authors knowledge, but it’s a great starting point. If scientists can dramatically shorten the time needed to generate the cells related to their particular disease of interest, then they can more quickly move on to the hard work ahead: gaining a deeper understanding of the disease and developing cures. Julian Gough, professor of bioinformatics at the University of Bristol and one of the senior researchers on the report, spoke of the potential impact of Mogrify in a university press release:

“The ability to produce numerous types of human cells will lead directly to tissue therapies of all kinds, to treat conditions from arthritis to macular degeneration, to heart disease. The fuller understanding, at the molecular level of cell production leading on from this, may allow us to grow whole organs from somebody’s own cells.”

 

Stem cell stories that caught our eye: reality check on chimeras, iPS cells for drug discovery and cell family history

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.

iPS cells becoming foot soldiers of drug discovery. Here at The Stem Cellar we write often about the power of iPS-type stem cells to model disease and accelerate drug development. This week provided a couple of strong reminders of the value of these induced pluripotent stem cells that researchers create by reprogramming any adult cell, usually skin or blood, into an embryonic stem cell-like state.

Researchers at Penn State University published work that used iPS cells from patients with Rett Syndrome to find a target for drug therapy for that severe form of autism spectrum disorder. After turning the stem cells into nerves they found those cells lacked a protein that is critical to the function of the neural transmitter GABA. That protein has now become a target for drug therapy. As a bonus for the field, the study, published in the Proceedings of the National Academy of Sciences, provided an explanation for why a drug already in clinical trials for Rett Syndrome might work. That drug is IGF1, insulin-like growth factor. The web site Medical News Today wrote up the research.

Later in the week an announcement popped up in my email for the two-day “inaugural” conference “Advances in iPS cell Technology for Drug Development Applications.” The field clearly has momentum. CIRM has funded a bank that will eventually house up to 3,000 cell lines relating to specific diseases. So far, 285 lines are available to researchers anywhere, 14 of them Autism spectrum lines, through the tissue banks at Coriell.

 

Tracking a cell’s family history. When cells divide their offspring can have a different identity from the mother cells. This occurs commonly in stem cells, as they mature into adult tissue, and in the immune system as cells respond to infections. Knowing the genetic details of how this happens could accelerate both stem cell science and our ability to understand and manipulate the immune system.

A team at MIT has taken us a step closer to this ability. They married a trendy new technique called single cell genetic analysis with a fluidic device that can isolate single cells in one chamber and daughter and grand daughter cells in subsequent chambers. In this case, they used single cell RNA-seq, the shorthand for sequencing. They wanted to know the differences between the cells in terms of genes that are actually active, and since the RNA representing a gene is only made when the gene is active, this provided a snapshot of each cell’s genetic identity.

Genetic Engineering News wrote about the work and quoted the lead author of the study Robert Kimmerling:

“Scientists have well-established methods for resolving diverse subsets of a population, but one thing that’s not very well worked out is how this diversity is generated. That’s the key question we were targeting: how a single founding cell gives rise to very diverse progeny.”

This new combined system should let researchers investigate how this happens. The MIT team started by looking at how one immune system cell can produce both the cells that attack and kill invaders and the cells that stick around and remember what the invaders looked like.

 

Human-animal chimeras, what are labs really doing. Antonio Regalado did a thorough piece in MIT Technology Review examining the work of the few labs around the country that are trying to grow human tissue in animal embryos—chimeras. He estimates that some 20 pig-human or sheep-human pregnancies have been established, but no one is letting those embryos grow more than a very few weeks. Their immediate goal is to better understand how the cells with different origins interact, not to breed chimeric animals.

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A pig at the UC Davis research center

One long-term goal is, for example, to grow a personalized new pancreas for diabetic patients who needs a new one of those insulin-producing organs. But no one in the field expects that to happen anytime soon. The process involves using modern genetic editing techniques to turn off the genes that would make a particular organ in the animal embryo, inserting human stem cells and hoping the growing embryo will hijack the genes for making the equivalent human organ, but not other human tissues.

The embryos examined so far have generally contained a very small amount of human DNA, less than one percent in a project at Stanford. So, probably not enough to give the animal human traits beyond the organ desired. Pablo Ross who has done some of the early work at the University of California, Davis explained the intent of those studies is “to determine the ideal conditions for generating human-animal chimeras.”

It is fascinating work and has great potential to alleviate organ shortages, but will require several more breakthroughs and much patience before that happens.

Stem cell stories that caught our eye: back repair, stem cell aging, babies for same sex couples, chimeras

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.

0c207-shutterstock_132771389Getting the right cells for back repair.  We often write that stem cells found in fat tissue can form bone, cartilage and other connective tissue. But that glosses over the fact that all those tissues come in many forms. A team in France has found a way to turn fat stem cells into the specific tissue found in the discs in our spine, that when deteriorated, leads to 40 percent of back pain.

The team at the French institute INSERM used two growth factors to turn stem cells from fat into cells called nucleus pulposus that make up the cushioning discs. Yahoo Finance ran a very short piece on the research that the team published in the journal Stem Cells.

 

Bone drug stops stem cell aging.  Doctors regularly use the drug zoledronate to improve the bone strength and extend the lives of patients with osteoporosis, but they don’t know exactly how it works. A team at the University of Sheffield in the U.K. pegged the drug’s role on reducing the natural DNA damage that occurs in the stem cells that build new bone. The drug in essence slows the aging of bone-forming stem cells.

They published their work in the journal Stem Cells and Health Medicine Network posted the university press release in which the authors speculate that the drug might have a similar impact on stem cells that are supposed to repair other tissues as well.

 “The drug enhances the repair of the damage in DNA occurring with age in stem cells in the bone. It is also likely to work in other stem cells too.”

The researchers suggest the drug could have a role in treating heart disease, muscle diseases and other age-related conditions.

 

Babies for same sex couples—not yet.  One recent journal publication contained no new scientific research yet generated many headlines, which often suggested an advance had been made that could allow same sex couples to have a completely biologically related baby. The original article, a review in the Journal of Law and the Biosciences, provided a look at the current state of the science of creating eggs and sperm from stem cells and an analysis of the ethical and policy implications of the work.

While the work is fairly advanced in mice, it is at very early stages in humans. Written by Sonia Suter of George Washington University in Washington, D.C., the review outlines potential benefits and risks of the process called in vitro gametogenesis (IVG). Medical News Today wrote a story from the university press release that quoted Suter:

“The ethical dilemmas about when and how such research should be done will be enormously challenging.”

Despite the many concerns, she suggests that with the strong support for other forms of assisted reproduction, eventually, IVG could be just another routine way to have a baby.

 

Chimeras can tell us about early development.  Another study that got a lot of press for the wrong reasons generated mouse embryos that contained tissue from human stem cells.  Those chimeras—organisms with cells from two species—were reported to prove the safety of pluripotent stem cells, those cells that can become any tissue in the body.

 

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Human cells (green) in a mouse embryo

The team placed human pluripotent stem cells, either reprogrammed iPS cells or embryonic stem cells, into early stage mouse embryos and saw them correctly turn into the three so-called germ layers that make up all parts of the body.  They only observed the tissue develop for two days, but during that time they saw no indication of tumors or inappropriate cell development.

However, as CIRM grantee Paul Knoepfler wrote in his The Niche blog the behavior of pluripotent stem cells in an early embryo, where you want them to behave like embryonic tissue, may not be relevant to how those cells would behave in an adult patient where it could be disastrous if the cells behaved in an embryonic fashion.

What the work by Victoria Mascetti and Roger Pederson of the University of Cambridge in the U.K. does provide is an elegant new tool to study early human embryonic development.

“Our finding that human stem cells integrate and develop normally in the mouse embryo will allow us to study aspects of human development during a window in time that would otherwise be inaccessible,” Mascetti said in a press release quoted in an article in The Scientist.

Four Challenges to Making the Best Stem Cell Models for Brain Diseases

Neurological diseases are complicated. A single genetic mutation causes some, while multiple genetic and environmental factors cause others. Also, within a single neurological disease, patients can experience varying symptoms and degrees of disease severity.

And you can’t just open up the brain and poke around to see what’s causing the problem in living patients. It’s also hard to predict when someone is going to get sick until it’s already too late.

To combat these obstacles, scientists are creating clinically relevant human stem cells in the lab to capture the development of brain diseases and the differences in their severity. However, how to generate the best and most useful stem cell “models” of disease is a pressing question facing the field.

Current state of stem cell models for brain diseases

Cold Spring Harbor Lab, Hillside Campus, Location: Cold Spring Harbor, New York, Architect: Centerbrook Architects

Cold Spring Harbor Lab, Hillside Campus, Location: Cold Spring Harbor, New York, Architect: Centerbrook Architects

A group of expert stem cell scientists met earlier this year at Cold Spring Harbor in New York to discuss the current state and challenges facing the development of stem cell-based models for neurological diseases. The meeting highlighted case studies of recent advances in using patient-specific human induced pluripotent stem cells (iPS cells) to model a breadth of neurological and psychiatric diseases causes and patient symptoms aren’t fully represented in existing human cell models and mouse models.

The point of the meeting was to identify what stem cell models have been developed thus far, how successful or lacking they are, and what needs to be improved to generate models that truly mimic human brain diseases. For a full summary of what was discussed, you can read a Meeting Report about the conference in Stem Cell Reports.

What needs to be done

After reading the report, it was clear that scientists need to address four major issues before the field of patient-specific stem cell modeling for brain disorders can advance to therapeutic and clinical applications.

1. Define the different states of brain cells: The authors of the report emphasized that there needs to be a consensus on defining different cell states in the brain. For instance, in this blog we frequently refer to pluripotent stem cells and neural (brain) stem cells as a single type of cell. But in reality, both pluripotent and brain stem cells have different states, which are reflected by their ability to turn into different types of cells and activate a different set of genes. The question the authors raised was what starting cell types should be used to model specific brain disorders and how do we make them from iPS cells in a reproducible and efficient fashion?

2. Make stem cell models more complex: The second point was that iPS cell-based models need to get with the times. Just like how most action-packed or animated movies come in 3D IMAX, stem cell models also need to go 3D. The brain is comprised of an integrated network of neurons and glial support cells, and this complex environment can’t be replicated on the flat surface of a petri dish.

Advances in generating organoids (which are mini organs made from iPS cells that develop similar structures and cell types to the actual organ) look promising for modeling brain disease, but the authors admit that it’s far from a perfect science. Currently, organoids are most useful for modeling brain development and diseases like microencephaly, which occurs in infants and is caused by abnormal brain development before or after birth. For more complex neurological diseases, organoid technology hasn’t progressed to the point of providing consistent or accurate modeling.

The authors concluded:

“A next step for human iPS cell-based models of brain disorders will be building neural complexity in vitro, incorporating cell types and 3D organization to achieve network- and circuit-level structures. As the level of cellular complexity increases, new dimensions of modeling will emerge, and modeling neurological diseases that have a more complex etiology will be accessible.”

3. Address current issues in stem cell modeling: The third issue mentioned was that of human mosaicism. If you think that all the cells in your body have the same genetic blue print, then you’re wrong. The authors pointed out that as many as 30% of your skin cells have differences in their DNA structure or DNA sequences. Remember that iPS cell lines are derived from a single patient skin or other cell, so the problem is that studies might need to develop multiple iPS cell lines to truly model the disease.

Additionally, some brain diseases are caused by epigenetic factors, which modify the structure of your DNA rather than the genetic sequence itself. These changes can turn genes on and off, and they are unfortunately hard to reproduce accurately when reprogramming iPS cells from patient adult cells.

4. Improve stem cell models for drug discovery: Lastly, the authors addressed the use of iPS cell-based modeling for drug discovery. Currently, different strategies are being employed by academia and industry, both with their pros and cons.

Industry is pursuing high throughput screening of large drug libraries against known disease targets using industry standard stem cell lines. In contrast, academics are pursuing candidate drug screening on a much smaller scale but using more relevant, patient specific stem cell models.

The authors point out that, “a major goal in the still nascent human stem cell field is to utilize improved cell-based assays in the service of small-molecule therapeutics discovery and virtual early-phase clinical trials.”

While in the past, the paths that academia and industry have taken to reach this goal were different, the authors predict a convergence between the paths:

“Now, research strategies are converging, and both types of researchers are moving toward human iPS cell-based screening platforms, drifting toward a hybrid model… New collaborations between academic and pharma researchers promise a future of parallel screening for both targets and phenotypes.”

Conclusions and Looking to the Future

This meeting successfully described the current landscape of iPS cell-based disease modeling for brain disorders and laid out a roadmap for advancing these stem cell models to a stage where they are more effective for understanding the mechanisms behind disease and for therapeutic screening.

I agree with the authors conclusion that:

“Moving forward, a critical application of human iPS cell-based studies will be in providing a platform for defining the cellular, molecular, and genetic mechanisms of disease risk, which will be an essential first step toward target discovery.”

My favorite points in the report were about the need for more collaboration between academia and industry and also the push for reproducibility of these iPS cell models. Ultimately, the goal is to understand what causes neurological disease, and what drugs or stem cell therapies can be used to cure them. While iPS cell models for brain diseases still have a way to go before being more clinically relevant, they will surely play a prominent role in attaining this goal.

Meeting Attendees

Meeting Attendees

Wiping out a cell’s identity shifts cellular reprogramming into high gear

Blog CAF-1 chromatin

The packaging of DNA into chromatin (image credit: Felsenfeld and Groudine, Nature 2013

If stretched out end to end, the DNA in just one cell of your body would reach a whopping six feet in length. A complex cellular structure called chromatin – made up of coils upon coils of DNA and protein – makes it possible to fit all that DNA into a single cell nucleus that’s only 0.0002 inches in diameter.

Chromatin: more than meets the eye
Once thought to merely play a structural role, mounds of data have shown that chromatin is also a critical regulator of gene activity. In fact, it’s a key component to maintaining a cell’s identity. So, for example, in the nucleus of a skin cell, genes related to skin function tend to lie within stretches of DNA having a loosely coiled chromatin structure. This placement makes the skin-related genes physically more accessible to become activated. But genes related to, say, heart, liver or brain cell function in that same skin cell tend to remain silent within tightly packaged, inaccessible chromatin.

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Depiction of (a) loosely packaged, accessible chromatin (red is DNA; blue is protein) vs (b) tightly packaged inaccessible chromatin. (Image credit: Interface Focus (2012) 2, 546–554)

As that skin cell divides and its DNA is replicated, there are various proteins that assemble and maintain the same chromatin positioning in their daughter cells, which helps them know they are skin cells. This cellular memory isn’t easy to erase, and it’s one of the reasons for the low efficiency when reprogramming a skin cell back into an embryonic stem cell-like state, also known as the induced pluripotent stem cell (iPSC) technique.

Blocking a DNA roadblock increases iPSC efficiency
So researchers at Harvard and in Vienna asked what if you blocked proteins responsible for arranging the chromatin – would it make it easier to generate iPSCs? The answer is a resounding “yes” based on data reported last Thursday in Nature. While previous studies asking the very same question have shown decent increases in iPSC reprogramming efficiency, this current research achieved orders of magnitude higher efficiency.

Using two independent screening methods, the research team systematically blocked the activity of hundreds of genes that play a role in the packaging of chromatin structure and maintaining cellular memory. These inhibition experiments were carried out in skin cells that were in the process of being reprogrammed into iPSCs. In both screening approaches, the inhibition of two proteins, collectively called chromatin assembly factor 1 (CAF-1), led to large increases in reprogramming efficiency.

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Induced pluripotent stem cell (iPS cell) colonies were generated after researchers at Harvard Stem Cell Institute suppressed the CAF1 gene. (Image credit: Sihem Chaloufi)

Inhibiting CAF-1 potently erases cell memory
While the inhibition of genes previously identified to block reprogramming led to a three to four-fold increase in iPSC generation, inhibition of CAF-1 dramatically increased efficiency 50 to 200 fold. Also, compared to a typical reprogramming time of nine days, in skin cells with CAF-1 inhibition, the first iPSCs were observed in just four days.

The increased ease of manipulating cells also applies to direct reprogramming. This alternative reprogramming method skips the iPSC process altogether and instead directly converts one adult cell type into another. In this case, the researchers were able to convert skin cells into neurons and one immune system cell type (B cells) into another (macrophages).

In a Harvard press release posted on Monday, co-first author Sihem Chaloufi, a postdoc in Konrad Hochedlinger’s lab at Harvard, succinctly described the overall finding:

“The cells forget who they are, making it easier to trick them into becoming another type of cell.”

This potent erasing of cell memory via CAF-1 inhibition could make it easier to derive many different cells types from iPSCs or direct reprogramming for use in drug testing, modeling human disease in a lab dish as well as scaling up production of future cell therapies.