Reprogramming cells with a nanochip, electricity and DNA to help the body to heal itself

The axolotl, a member of the salamander family, has amazing regenerative abilities. You can cut off its limbs or crush its spinal cord and it will repair itself with no scarring. A human’s healing powers, of course, are much more limited.

To get around this unfortunate fact, the field of regenerative medicine aims to develop stem cell-based therapies that provide the body with that extra oomph of regenerative ability to rid itself of disease or injury. But most of the current approaches in development rely on complex and expensive manufacturing processes in clinical labs before the cells can be safely transplanted in a patient’s body. Wouldn’t it be nice if we could just give the cells already in our bodies some sort of spark to allow them to repair other diseased or damaged cells?

A research team at Ohio State University have taken a fascinating step toward that seemingly science fiction scenario. Reporting this week in Nature Nanotechnology, the scientists describe a technique that – with some DNA, a nanochip and an electric current placed on the skin – can help mice regrow blood vessels to restore dying tissue.

Researchers demonstrate a process known as tissue nanotransfection (TNT). In laboratory tests, this process was able to heal the badly injured legs of mice in just three weeks with a single touch of this chip. The technology works by converting normal skin cells into vascular cells, which helped heal the wounds. Photo: Wexner Medical Center/The Ohio State University

The foundation of this technique is cellular reprogramming. Induced pluripotent stem cells are the most well-known example of reprogramming in which adult cells, like skin or blood, are converted, in a lab dish, to an embryonic stem cell-like state by introducing a set of reprogramming genes into the cells. From there, the stem cells can be specialized into any cell type.

Now, you wouldn’t want to convert skin or blood cells inside the body into quasi embryonic stem cells because they could generate tumors due to their limitless ability to multiply. In this study, the researchers rely on a related method, direct reprogramming, that skips the stem cell step and uses a different set of genes to directly convert one cell type into another. They focused on the direct reprogramming of skin cells to endothelial cells, a key component of blood vessels, in mice that were given symptoms mimicking those seen in human injury-induced limb ischemia. This condition leads to a risk of gangrene and amputation when severely injured limbs deteriorate due to blocked blood vessels.

It’s one thing to introduce, or transfect, reprogramming genes into cells that are grown in the very controlled environment of a petri dish. But how the heck does one get DNA into skin cells on the leg of a mouse? That’s where the team’s tissue nano-transfection (TNT) approach comes into the picture. After rubbing off a small section of dead skin on the leg, the TNT device, composed of an nanochip electrode and tiny channels of liquid containing reprogramming DNA, is placed on the skin. A short pulse of electricity is applied which opens miniscule holes in the membranes of skin cells that are in contact with the electrode which allows the DNA to enter the cells. Here’s a short video describing the process:

Three weeks after the procedure, blood vessels had formed, blood flow was restored and the legs of the mice were saved. Team leader, Dr. Chandan Sen, described the results in an interview with National Public Radio:

“Not only did we make new cells, but those cells reorganized to make functional blood vessels that plumb with the existing vasculature and carry blood.”

It’s surprising that TNT reprogramming affects more than just the skin cells that were in contact with the device. But it appears the reprogramming instructions from the introduced DNA was somehow spread to other cells through tiny vesicles called exosomes. When Sen’s team extracted those exosomes and introduced them to skin cells in a petri dish, those cells specialized into blood vessel cells.

This result did attract some skepticism from the field. In the NPR story, stem cell expert Dr. Sean Morrison had this to say:

“There are all manners of claims of these vesicles. It’s not clear what these things are, and if it’s a real biological process or if it’s debris.”

Clearly, more work is needed before TNT is ready for clinical trials in humans. But if it holds up, the technique could bring us closer to the incredible self-healing powers of the axolotl.

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Could the Answer to Treating Parkinson’s Disease Come From Within the Brain?

Sometimes a solution to a disease doesn’t come in the form of a drug or a stem cell therapy, but from within ourselves.

Yesterday, scientists from the Karolinska Institutet in Sweden reported an alternative strategy for treating Parkinson’s disease that involves reprogramming specific cells in the brain into the nerve cells killed off by the disease. Their method, which involves delivering reprogramming genes into brain cells called astrocytes, was able to alleviate motor symptoms associated with Parkinson’s disease in mice.

What is Parkinson’s Disease and how is it treated?

Parkinson’s disease (PD) is a progressive neurodegenerative disease that’s characterized by the death of dopamine-producing nerve cells (called dopaminergic neurons) in an area of the brain that controls movement.

Dopaminergic neurons grown in a culture dish. (Image courtesy of Faria Zafar, Parkinson’s Institute).

PD patients experience tremors in their hands, arms and legs, have trouble starting and stopping movement, struggle with maintaining balance and have issues with muscle stiffness. These troublesome symptoms are caused by a lack dopamine, a chemical made by dopaminergic neurons, which signals to the part of the brain that controls how a person initiates and coordinates movement.

Over 10 million people in the world are affected by PD and current therapies only treat the symptoms of the disease rather than prevent its progression. Many of these treatments involve drugs that replace the lost dopamine in the brain, but these drugs lose their effectiveness over time as the disease kills off more neurons, and they come with their own set of side effects.

Another strategy for treating Parkinson’s is replacing the lost dopaminergic neurons through cell-based therapies. However this research is still in its early stages and would require patients to undergo immunosuppressive therapy because the stem cell transplants would likely be allogeneic (from a donor) rather than autologous (from the same individual).

Drug and cell-based therapies both involve taking something outside the body and putting it in, hoping that it does the right thing and prevents the disease. But what about using what’s already inside the human body to fight off PD?

This brings us to today’s study where scientists reprogrammed brain cells in vivo (meaning inside a living organism) to produce dopamine in mice with symptoms that mimic Parkinson’s. Their method, which was published in the journal Nature Biotechnology, was successful in alleviating some of the Parkinson’s-related movement problems the mice had. This study was funded in part by a CIRM grant and received a healthy amount of coverage in the media including STATnews, San Diego Union-Tribune and Scientific American.

Reprogramming the brain to make more dopamine

Since Shinya Yamanaka published his seminal paper on reprogramming adult somatic cells into induced pluripotent stem cells, scientists have taken the building blocks of his technology a step further to reprogram one adult cell type into another. This process is called “direct reprogramming” or “transdifferentiation”. It involves delivering a specific cocktail of genes into cells that rewrite the cells identity, effectively turning them into the cell type desired.

The Karolinska team found that three genes: NEUROD1, ASCL1 and LMX1A combined with a microRNA miR218 were able to reprogram human astrocytes into induced dopaminergic neurons (iDANs) in a lab dish. These neurons looked and acted like the real thing and gave the scientists hope that this combination of factors could reprogram astrocytes into iDANs in the brain.

The next step was to test these factors in mice with Parkinson’s disease. These mice were treated with a drug that killed off their dopaminergic neurons giving them Parkinson’s-like symptoms. The team used viruses to deliver the reprogramming cocktail to astrocytes in the brain. After a few weeks, the scientists observed that some of the “infected” astrocytes developed into iDANs and these newly reprogrammed neurons functioned properly, and more importantly, helped reverse some of the motor symptoms observed in these mice.

This study offers a new potential way to treat Parkinson’s by reprogramming cells in the brain into the neurons that are lost to the disease. While this research is still in its infancy, the scientists plan to improve the safety of their technology so that it can eventually be tested in humans.

Bonus Blog Interview for World Parkinson’s Day

Ernest Arenas, Karolinska Institutet

In honor of World Parkinson’s day (April 11th), I’m providing a bonus blog interview about this research. I reached out to the senior author of this study, Dr. Ernest Arenas, to ask him a few more questions about his publication and the future studies his team is planning.

Q) What are the major findings of your current study and how do they advance research on Parkinson’s disease?

The current treatment for Parkinson’s disease (PD) is symptomatic and does not change the course of the disease. Cell replacement therapies, such as direct in vivo reprogramming of in situ [local] astrocytes into dopamine (DA) neurons, work by substituting the cells lost by disease and have the potential to halt or even reverse motor alterations in PD.

Q) Can you comment on the potential for gene therapy treatments for Parkinson’s patients?

We see direct in vivo reprogramming of brain astrocytes into dopamine neurons in situ as a possible future alternative to DA cell transplantation. This method represents a gene therapy approach to cell replacement since we use a virus to deliver four reprogramming factors. In this method, the donor cells are in the host brain and there is no need to search for donor cells and no cell transplantation or immunosuppression. The method for the moment is an experimental prototype and much more needs to be done in order to improve efficiency, safety and to translate it to humans.

Q) Will reprogrammed iDANs be susceptible to Parkinson’s disease over time?

As any other cell replacement therapy, the cells would be, in principle, susceptible to Parkinson’s disease. It has been found that PD catches up with transplanted cells in 15-20 years. We think that this is a sufficiently long therapeutic window.

In addition, direct in vivo reprogramming may also be performed with drug-inducible constructs that could be activated years after, as disease progresses. This might allow adding more cells by turning on the reprogramming factors with pharmacological treatment to the host. This was not tested in our study but the basic technology to develop such strategies currently exist.

Q) What are your plans for future studies and translating this research towards the clinic?

In our experiments, we used transgenic mice in order to test our approach and to ensure that we only reprogrammed astrocytes. There is a lot that still needs to be done in order to develop this approach as a therapy for Parkinson’s disease. This includes improving the efficiency and the safety of the method, as well as developing a strategy suitable for therapy in humans. This can be achieved by further improving the reprogramming cocktail, by using a virus with a selective tropism [affinity] for astrocytes and that do not incorporate the constructs into the DNA of the host cell, as well as using constructs with astrocyte-specific promoters and capable of self-regulating depending on the cell context.

Our study demonstrates for the first time that it is possible to use direct reprogramming of host brain cells in order to rescue neurological symptoms. These results indicate that direct reprogramming has the potential to become a novel therapeutic approach for Parkinson’s disease and opens new opportunities for the treatment of patients with neurological disorders.

CIRM-funded team uncovers novel function for protein linked to autism and schizophrenia

Imagine you’ve just stopped your car at the top of the steepest street in San Francisco. Now, if want to stay at the top of the hill you’re going to need to keep your foot on the brakes. Let go and you’ll start rolling down. Fast.

Don’t step off the brake pedal! Photo: Wikipedia

Conceptually, similar decision points happen in human development. A brain cell, for instance, has the DNA instructions to become any cell in the body but must “keep the brakes on”, or repress, genes responsible for other cell types. Release the silencing of those genes and the brain cell’s properties will get pulled toward other fates.

That’s the subject of a CIRM-funded research study published today in Nature which reports on the identification of a new type of repressor protein which opens up a new understanding of how brain cells establish and keep their identity. That may not sound so exciting to our non-scientist readers but this discovery could lead to new therapy approaches for neurological disorders like autism, schizophrenia, major depression and low I.Q.

Skin cells to brain cells with just three genes
In previous experiments, this Stanford University research team led by Marius Wernig, showed it’s possible to convert a skin cell to a brain cell, or neuron, by adding just three genes to the cells, including one called Myt1l. The other two genes were known to act as master “on switches” that activate a cascade of genes responsible for making neuron-specific proteins. Myt1l also helped increase the efficiency of this direct reprogramming but it’s exact role in the process wasn’t clear.

Direct conversion of skin cell into a neuron.
Image: Wernig Lab, Stanford

A closer examination of Myt1l protein function revealed that instead of being an on switch for neuron-specific genes, it was actually an off switch for skin-specific genes. Now, there’s nothing unusual about the existence of a protein that represses gene activity to help determine cell identity. But up until now, these repressors were thought to be “lineage specific” meaning they specifically switched off genes of a specific cell type. For example, a well-studied repressor called REST affects cell fate by putting the brakes on only nerve-specific genes. The case of Myt1l was different.

Many but one
The researchers found that, in brain cells, Myt1l not only blocked the activation of skin-specific genes, it also shut down genes related to lung, cartilage, heart and other cells fates. The one set of genes that Mytl1 repressor did not appear to act on was neuron-specific genes. From these results a “many but one” pattern emerged. That is; it seems Myt1l helps drive and maintain a neuron cell fate by shutting off gene networks for many different cell identities except for neurons. It’s a novel way to regulate cell fate, as Wernig explained in a press release:

Marius Wernig
Photo: Steve Fisch

“The concept of an inverse master regulator, one that represses many different developmental programs rather than activating a single program, is a unique way to control neuronal cell identity, and a completely new paradigm as to how cells maintain their cell fate throughout an organism’s lifetime.”

To build a stronger case for Myt1l function, the team looked at the effect of blocking the protein in the developing mouse brain. Sure enough, lifting Myt1l repression lead to a decrease in the number of neurons in the brain. Wernig described the impact of also inhibiting Myt1l in mature neurons:

“When this protein is missing, neural cells get a little confused. They become less efficient at transmitting nerve signals and begin to express genes associated with other cell fates.”

Potential cures can be uncovered withfundamental lab research
It turns out that Myt1l mutations have been recently found in people with autism, schizophrenia, major depression and low I.Q. Based on their new insights, the author suggest that in adults, these disorders may be caused by a neuron’s inability to maintain its identity rather than by a more permanent abnormality that occurred during fetal brain development. This hypothesis presents the exciting possibility of developing therapies that could improve symptoms.

A new and improved method for making healthy heart tissue is here

Scientists from the Gladstone Institutes have done it again. They’ve made a better and faster way of generating healthy heart tissue in mice with damaged hearts. With further advancements, their findings could potentially be translated into a new way of treating heart failure in patients.

Previously, the Gladstone team discovered that they could transform scar tissue in the damaged hearts of mice into healthy, beating heart muscle cells by a process called direct reprogramming. The team found that turning on three transcription factors, Gata4, Mef2c and Tbx5 (collectively called GMT), in the damaged hearts of mice activated heart genes that turned scar tissue cells, also known as cardiac fibroblasts, into beating heart cells or cardiomyocytes.

Their GMT direct cardiac reprogramming technology was only able to turn 10 percent of cardiac fibroblasts into cardiomyocytes in mice over the period of six to eight week. In their new CIRM-funded study published in Circulation, they improved upon their original reprogramming method by identifying two chemicals that improved the efficiency of making new heart cells. Not only were they able to create eight times the number of beating cardiomyocytes from mouse cardiac fibroblasts, but they were also able to speed up the reprogramming process to a period of just one week.

To find these chemicals, they screened a library of 5,500 small molecules. The chemicals that looked most promising for cardiac reprogramming were inhibitors of the TGF-β and WNT signaling pathways. The importance of these chemicals was explained in a Gladstone news release:

“The first chemical inhibits a growth factor that helps cells grow and divide and is important for repairing tissue after injury. The second chemical inhibits an important pathway that regulates heart development. By combining the two chemicals with GMT, the researchers successfully regenerated heart muscle and greatly improved heart function in mice that had suffered a heart attack.”

Senior author on the study, Deepak Srivastava, further explained:

“While our original process for direct cardiac reprogramming with GMT has been promising, it could be more efficient. With our screen, we discovered that chemically inhibiting two biological pathways active in embryonic formation improves the speed, quantity, and quality of the heart cells produced from our original process.”

Encouraged by their studies in mice, the scientists also tested their new and improved direct reprogramming method on human cells. Previously they found that while the same GMT transcription factors could reprogram human cardiac fibroblasts into cardiomyocytes, a combination of seven factors was required to make quality cardiomyocytes comparable to those seen in mice. But with the addition of the two inhibitors, they were able to reduce the number of reprogramming factors from seven to four, which included the GMT factors and one additional factor called Myocardin. These four factors plus the two chemical inhibitors were capable of reprograming human cardiac fibroblasts into beating heart cells.

With heart failure affecting more than 20 million people globally, the need for new therapies that can regenerate the heart is pressing. The Gladstone team is hoping to advance their research to a point where it could be tested in human patients with heart failure. First author on the study, Tamer Mohamed, concluded:

“Heart failure afflicts many people worldwide, and we still do not have an effective treatment for patients suffering from this disease. With our enhanced method of direct cardiac reprogramming, we hope to combine gene therapy with drugs to create better treatments for patients suffering from this devastating disease.”

Tamer Mohamed and Deepak Srivastava, Gladstone Institutes

Tamer Mohamed and Deepak Srivastava. Photo courtesy of Chris Goodfellow, Gladstone Institutes


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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.”

 

A Win for Diabetes: Scientists Make Functional Pancreatic Cells From Skin

Today is an exciting day for diabetes research and patients. For the first time, scientists have succeeded in making functional pancreatic beta cells from human skin. This new method for making the insulin-producing cells of the pancreas could produce a new, more effective treatment for patients suffering from diabetes.

Researchers at the Gladstone Institutes and the University of California, San Francisco published these promising findings today in the journal Nature Communications.

Making pancreatic cells from skin

They used a technique called direct reprogramming to turn human skin cells directly into pancreatic beta cells without having to go all the way back to a pluripotent stem cell state. The skin cells were treated with factors used to generate induced pluripotent stem cells (iPSCs) and with pancreatic-specific molecules. This cocktail of factors and molecules shut off the skin genes and turned on genes of the pancreas.

The end product was endoderm progenitor cells, which are like stem cells but can only generate cell types specific to organs derived from the endoderm layer (for example: lungs, thyroid, pancreas). The scientists took these endoderm progenitors and further coaxed them into mature, pancreatic beta cells after treatment with another cocktail of molecules.

Functioning human pancreatic cells after they’ve been transplanted into a mouse. (Image: Saiyong Zhu, Gladstone)

Functioning human pancreatic cells after they’ve been transplanted into a mouse. (Image: Saiyong Zhu, Gladstone)

While the pancreatic cells they made looked and acted like the real thing in a dish (they were able to secrete insulin when exposed to glucose), the authors needed to confirm that they functioned properly in animals. They transplanted the mature beta cells into mice that were engineered to have diabetes, and observed that the human beta cells protected the mice from becoming diabetic by properly regulating their blood glucose levels.

Importantly, none of the mice receiving human cells got tumors, which is always a concern when transplanting reprogrammed cells or cells derived from pluripotent stem cells.

What does this mean?

This study is groundbreaking because it offers a new and more efficient method to make functioning human beta cells in mass quantities.

Dr. Sheng Ding, a CIRM funded senior investigator at the Gladstone and co-senior author, explained in a Gladstone news release:

Sheng Ding

Sheng Ding

“This new cellular reprogramming and expansion paradigm is more sustainable and scalable than previous methods. Using this approach, cell production can be massively increased while maintaining quality control at multiple steps. This development ensures much greater regulation in the manufacturing process of new cells. Now we can generate virtually unlimited numbers of patient-matched insulin-producing pancreatic cells.”

 

Matthias Hebrok, director of the Diabetes Center at UCSF and co-senior author on paper discussed the potential research and clinical applications of their findings:

Mattias Hebrok

Matthias Hebrok

“Our results demonstrate for the first time that human adult skin cells can be used to efficiently and rapidly generate functional pancreatic cells that behave similar to human beta cells. This finding opens up the opportunity for the analysis of patient-specific pancreatic beta cell properties and the optimization of cell therapy approaches.”

 

The study does mention the caveat that their direct reprogramming approach wasn’t able to generate all the cell types of the pancreas. Having these support cells would better recreate the pancreatic environment and likely improve the function of the transplanted beta cells.

Lastly, I find this study exciting because it kills two birds with one stone. Scientists can use this technique to make better cellular models of diabetes to understand why the disease happens, and they could also develop new cell replacement therapies in humans. Already, stem cell derived pancreatic beta cells are being tested in human clinical trials for type 1 diabetes (one of them is a CIRM-funded clinical trial by Viacyte) and it seems likely that beta cells derived from skin will follow suit.


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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.

Chromatin_open_closed

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.

Blog CAF -1 105305_web

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.

Bringing down the gatekeeper for a stem cell-based Parkinson’s cure

Feng-dopamine-HI

University of Buffalo researchers converted these dopamine neurons directly from human skin cells. Image shows a protein found only in neurons (red) and an enzyme that synthesizes dopamine (green). Cell DNA is labeled in blue.

On the surface, a stem cell-based cure for Parkinson’s disease seems pretty straight-forward. This age-related neurodegenerative disorder, which leads to progressively worsening tremors, slowness of movement and muscle rigidity, is caused by the death of a specific type of nerve cell, or neuron, that produces the chemical dopamine in a specific region of the brain. So it would seem that simply transplanting stem cell-derived dopamine-producing neurons (DA neurons) in the brains of Parkinson’s patients to replace the lost cells would restore dopamine levels and alleviate Parkinson’s symptoms.

Easier said than done
Well, it hasn’t turned out to be that easy. After initial promising results using fetal brain stem cell transplants in the 80’s and 90’s, larger clinical trials showed no significant benefit and even led to a worsening of symptoms in some patients. One potential issue with those early trials could have been variable cell composition of the fetal cell-based therapy. On top of that, the availability of fetal tissue is limited and the quantities of transplantable cells obtained from these samples are very low.

More recently, researchers have been busy at generating more pure populations of DA neurons from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). Great progress has been made so far, but the field is still hampered by not being able to make enough DA neurons from hESCs and iPSCs in a timely manner.

Cutting out the pluripotent “middle man”
This week a research team at the University of Buffalo reported in Nature Communications about a much more efficient method for producing DA neurons. It’s a finding that could provide a strong push towards stem cell-based therapy development for Parkinson’s disease.

The team bypassed the need to start with hESCs or iPSCs and instead converted skin cells directly into DA neurons. A thorough analysis of the cells confirmed that they were functional and matched that characteristics of the specific dopamine neurons that are lost in Parkinson’s.

This direct reprogramming of skin cells into DA neurons as well as other cells is a technique pioneered by several independent researchers including some of our own grantees. This method is thought to have a few advantages over the specialization of immature hESCs or iPSCs into tissue-specific cells. Not only is the direct reprogramming process faster it also doesn’t require cell division so there’s less concern about the introduction of DNA mutations and the potential of tumor formation. Another plus for direct reprogramming is the possibility of inducing the direct conversion of one cell type into another inside the body rather than relying on the manipulation of hESCs and iPSCs in the lab. Still, despite these advantages the efficiency of direct reprogramming is still very low. That’s where the University of Buffalo team comes into the picture.

Bringing down the gatekeeper
The researchers led by physiology and biophysics professor Jian Feng, made a few key modifications to increase the efficiency of the current skin cell to DA neuron direct reprogramming methods. They first reduced the level of a protein call p53. This protein has several nicknames like “guardian of our genes” and “tumor suppressor” because it plays critical roles in controlling cell division and DNA repair and, in turn, helps keep a clamp on cell growth.

Reducing the presence of p53 during the direct reprogramming process led to a much more efficient conversion of skin cells to DA neurons. And because the conversion from a skin cell to a neuron happens quickly – just a couple days – timing the introduction of cell nutrients specific to neurons had to be carefully watched. Together, these tweaks improved upon previous studies as Feng mentioned in a University of Buffalo press release:

“The best previous method could take two weeks to produce 5 percent dopamine neurons. With ours, we got 60 percent dopamine neurons in ten days.”

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Jian Feng, PhD, professor of physiology and biophysics, Jacobs School of Medicine and Biomedical Sciences, University of Buffalo

IMHO (In my humble opinion)
I imagine there’s a lot more work ahead to get this method of deriving DA neurons from skin ready for the clinic. This reprogramming technique relied on the introduction of neuron-specific genes into the skin cells using a deactivated virus as the means of delivery. Even though the virus is inactive, its viral DNA randomly inserts into the cells’ chromosomes which can turn on genes that cause cancer. Therefore, a non-viral version of this method would need to be developed for clinical use.

Also, as mentioned earlier, since p53 inhibits tumors by suppressing uncontrolled cell division, it would be important to make sure that a reduction of p53 didn’t lead to any long-term negative consequences, like the transplantation of potentially cancerous cells into the patient.

Still, this dramatic increase in efficiency for making functional DA neurons and the identification of p53 as a key control point for direct reprogramming are very exciting developments for a disease field that is committed to finding cures for its patients.

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From the Stem Cellar archives: blogs about direct reprogramming
Video: CIRM Grantee Marius Wernig discusses direct reprogramming
Video: Thirty second elevator pitch describing direct reprogramming

Skipping a Step: Turning Brain Cells Directly into Neurons

It was once commonly believed that “what you see is what you get” with the human brain. As in, the brains cells that you are born with are the only ones you’ll have for the rest of your life because they can’t regenerate.

The discovery of brain stem cells in the late 90s disproved this notion and established that the brain can replace old cells and repair damage after injury. The brain’s regenerative capacity is limited, however. Consequently, patients suffering from neurodegenerative diseases like Alzheimer’s and Parkinson’s can’t rely on their brain stem cells to repopulate all of the sick and dying neurons in their brains.

This is where cellular reprogramming technology could come to the rescue. Induced pluripotent stem cells (iPSCs) generated from patient skin cells by cellular reprogramming can be turned into many types of brain cells to study diseases in a petri dish, as well as to test drugs and develop stem cell therapies. Eventually, the hope is to transplant iPSC-derived brain cells back into patient brains to treat or cure degenerative diseases.

Making neurons directly from other brain cells

Another form of cellular reprogramming, offers a more direct approach to generating populations of healthy brain cells. Using a similar technique to iPSCs, scientists can use specific factors to directly reprogram skin cells or other brain cells into neurons without making them go through the pluripotent stem cell state. By skipping a step in the reprogramming process, researchers save time, money, and energy – and it could result in safer cells.

The group used small molecules to directly reprogram human astrocytes into neurons that could be transplanted into mice. (Zhang et al., 2015)

The group used small molecules to directly reprogram human astrocytes into neurons that could be transplanted into mice. (Zhang et al., 2015)

While direct reprogramming of skin and non-neuronal brain cells into neurons has been published before, a study in Cell Stem Cell by a group at Penn State University last week described a new-and-improved method to make properly functioning neurons from brain astrocytes. Astrocytes are a type of glial cell that are abundant in the brain. They provide neurons with support, nutrients, and aid following injury.

Led by senior author Gong Chen, the group bathed human astrocytes in a cocktail of small molecules that turned the astrocytes into neurons in less than 10 days. These neurons survived in a dish for more than 5 months and were able to send electrical signals to each other (a sign that they were functional). Even more exciting was that the directly reprogrammed neurons survived and functioned properly when they were transplanted into the brains of mice.

When they studied the biological mechanism behind their direct reprogramming method, they found that the small molecule cocktail turned off the activity of astrocyte-specific genes in the astrocytes and turned on neuron-specific genes to convert them into neurons.

Human astrocytes (left) were directly reprogrammed into neurons (right). (Zhang et al., 2015)

Human astrocytes (left) were directly reprogrammed into neurons (right). (Zhang et al., 2015)

This discovery is great news for the reprogramming field as using small molecule reprogramming instead of the commonly used transcription-factor based reprogramming (which involves using viruses that can damage or alter the genome) is a more attractive method with broader applications­ for making neurons that can be transplanted into humans.

Direct reprogramming makes new neurons in the brain

But wait, there’s more! An article from TheScientist reported that multiple groups at the Society for Neuroscience (SFN) conference  in Chicago presented results on directly converting glial cells into neurons in mouse brains rather than in a dish.

One group from the Johannes Gutenberg University used two transcription factors, proteins that control which genes are turned on or off in the human genome, to directly reprogram mouse astrocytes into neurons. By producing more of Sox2 and Ascl1 in the cortex of the mouse brain than would normally be found there, they were able to turn 15% of the glial cells in that area into neurons.

The function of these directly reprogrammed neurons remains to be determined, but the lead scientist, Sophie Peron, told TheScientist:

“That’s the next step. Now that we have a system to get these cells converted we are currently studying their connectivity, functionality, and precise characteristics.”

Two other groups also reported similar findings when they worked with a type of glial cell called reactive astrocytes. These cells are specifically activated during injury to jumpstart the healing process. The first group from the University of Texas Southwestern used the factor Sox2 to directly reprogram reactive astrocytes into neurons in mice, while the group from Penn State University – mentioned earlier in this blog – did the same thing, but using a different factor, NeuroD1.

The Penn State group went further to test their direct reprogramming method in a mouse model of stroke and found that NeuroD1-reprogrammed neurons reduced cell death and tissue scarring after stroke.

Lead scientist Yuchen Chen said:

“These findings suggest that direct reprogramming of glial cells into functional neurons may provide a completely new approach for brain repair after stroke. Our next step is to analyze whether the glia-neuron conversion technology can facilitate functional recovery in stroke animals.”


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Keeping elderly cells old to understand the aging process

Aging is a key risk factor for many diseases, particularly disorders of the brain like Alzheimer’s or Parkinson’s, which primarily occur in the elderly. So a better understanding of the aging process should provide a better understanding of these neurodegenerative diseases.

The induced pluripotent stem cell (iPSC) technique makes it possible to grow human brain cells, or neurons, in the lab from elderly patient skin samples. Unfortunately, this method has a major pitfall when it comes to aging research: reprogramming skin cells back into the embryonic stem cell-like state of iPSCs strips away many of their old age-related characteristics.

Based on data published last week in Cell Stem Cell, Salk Institute researchers used a different technique called direct reprogramming as a means to keep old cells old. This alternative method sidesteps the need to make iPSCs (which brings cells all the way back to the pluripotent state) and instead converts a skin cell directly into the desired cell type.

First author Jerome Mertens and senior author Rusty Gage (Courtesy of the Salk Institute for Biological Studies).

First author Jerome Mertens and senior author Rusty Gage (Courtesy of the Salk Institute for Biological Studies).

iPSC and direct reprogramming go head-to-head

The study, funded in part by CIRM, relied on skin samples from people ranging in age from newly born to 89 years. The team generated iPSC and iPSC-derived neurons from these samples. They also made so-called induced neurons (iNs) from the skin cells using the direct reprogramming method. Other CIRM grantees have pioneered direct reprogramming of skin into nerve cells (see link below).

Skin cell samples from elderly human donors are directly converted into induced neurons (iNs), shown. (image: Courtesy of the Salk Institute for Biological Studies)

Skin cells from elderly human donors are directly converted into induced neurons (iNs), shown. (Image courtesy of the Salk Institute for Biological Studies).

When comparing skin cells from donors younger than 40 years old versus cells from the over 40 group, the team found several genes had age-dependent activity patterns. Those differences virtually disappeared in the iPSCs and iPSC-derived neurons from the same individuals. However, unlike iPSCs, direct reprogramming of the skin cells to neurons (iNs) hung on to age-dependent differences in gene activity.

Loss of RanBP17 protein a fountain of youth in reverse

A deeper analysis identified one gene called RanBP17 whose activity levels declined with increased age of the donor in both the original skin cells and those directly converted into iNs. But when those same donor skin cells were turned into iPSCs or even iPSC-derived neurons, RanBP17 levels in the older cells were no longer reduced and were on par with RanBP17 levels in the younger cells. In follow up experiments, a reduction in RanBP17 protein led to glitches in the transport of proteins into the cell’s nucleus, which other studies have linked to neurodegenerative diseases as well as the aging process.

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Gene expression patterns of age-related factors like RanBP17 are maintained in induced neurons but not iPSCs. (Mertens et al., 2015)

Altogether, these results encourage researchers to select iNs over iPSC-derived neurons when it comes to faithfully representing the aging process of brain cells. Based on a Salk Institute press release, you can tell that professor Martin Hezter, a contributing author, is excited about future studies with iNs:

By using this powerful approach, we can begin to answer many questions about the physiology and molecular machinery of human nerve cells–not just around healthy aging but pathological aging as well.

 


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