CIRM-funded scientists discover a new way to make stem cells using antibodies

Just as learning a new skill takes time to hone, scientific discoveries take time to perfect. Such is the case with induced pluripotent stem cells (iPSCs), the Nobel Prize winning technology that reprograms mature adult cells back into a pluripotent stem cell state. iPSCs are a powerful tool because they can develop into any cell found in the body. Scientists use iPSCs to model diseases in a dish, screen for new drugs, and to develop stem cell-based therapies for patients.

iPSCs grown in a cell culture dish.

The original iPSC technology, discovered by Dr. Shinya Yamanaka in 2006, requires viral delivery of four transcription factor genes, Oct4, Sox2, Klf4, and c-Myc, into the nucleus of an adult cell. These genes are inserted into the genome where they are activated to churn out their respective proteins. The combined expression of these four factors (OSKM) turns off the genetic programming of an adult cell and turns on the programming for a pluripotent stem cell.

The technology is pretty neat and allows scientists to make iPSCs from patients using a variety of different tissue sources including skin, blood, and even urine. However, there is a catch. Inserting reprogramming genes into a cell’s genome can be disruptive if the reprogramming genes fail to switch off or can cause cancer if nefarious oncogenes are turned on.

In response to this concern, scientists are developing alternative methods for making iPSCs using non-invasive methods. A CIRM-funded team from The Scripps Research Institute (TSRI) published such a study yesterday in the journal Nature Biotechnology.

Led by senior author and CIRM grantee Dr. Kristin Baldwin, the TSRI team screened a large library of antibodies – proteins that recognize and bind to specific molecules – to identify ones that could substitute for the OSKM reprogramming factors. The hope was that some of these antibodies would bind to proteins on the surface of cells and turn on a molecular signaling cascade from the outside that would turn on the appropriate reprogramming genes from the inside of the cell.

The scientists screened over 100 million antibodies and found ones that could replace three of the four reprogramming factors (Oct4, Sox2, and c-Myc) when reprogramming mouse skin cells into iPSCs. They were unable to find an antibody to replace Klf4 in the current study but have it on their to-do list for future studies.

Dr. Baldwin explained how her team’s findings improve upon previous reprogramming methods in a TSRI news release,

Kristen Baldwin

“This result suggests that ultimately we might be able to make IPSCs without putting anything in the cell nucleus, which potentially means that these stem cells will have fewer mutations and overall better properties.”

 

Other groups have published other non-invasive iPSC reprogramming methods using cocktails of chemicals, proteins or microRNAs in place of virally delivering genes to make iPSCs. However, Baldwin’s study is the first (to our knowledge) to use antibodies to achieve this feat.

An added benefit to antibody reprogramming is that the team was able to learn more about the signaling pathways that were naturally activated by the iPSC reprogramming antibodies.

“The scientists found that one of the Sox2-replacing antibodies binds to a protein on the cell membrane called Basp1. This binding event blocks Basp1’s normal activity and thus removes the restraints on WT1, a transcription factor protein that works in the cell nucleus. WT1, unleashed, then alters the activity of multiple genes, ultimately including Sox2’s, to promote the stem cell state using a different order of events than when using the original reprogramming factors.”

iPSCs made by antibody reprogramming could address some of the long-standing issues associated with more traditional reprogramming methods and could offer further insights into the complex signaling required to turn adult cells back into a pluripotent state. Baldwin and her team are now on the hunt for antibodies that will reprogram human (rather than mouse) cells into iPSCs. Stay tuned!

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New iPS Insights: Cell Damage Enhances Reprogramming

Researchers learn a ton about the biological function of cells by studying them in vitro; that is, outside the body in a petri dish. But inside the body, or in vivo, cells respond to surrounding proteins and other cells that may be missing in an in vitro experiment. Important insights waiting to be revealed can easily be overlooked if a cell isn’t analyzed in the right context.

That’s the lesson learned from a recent study in Science looking at the induced pluripotent stem (iPS) cell process of reprogramming adult skin cells into an embryonic stem cell-like state. By examining this technique in laboratory mice, a research team at the Spanish National Cancer Research Centre (CNIO) showed that, compared to isolated cells in vitro, the efficiency of in vivo cellular reprogramming in the mice is boosted by nearby damaged cells. So injured cells appear to provide a signal to help kick start the regenerative process.

(Watch this video for a quick recap of the report or read on for a few more details)

The history of iPS cells in 30 seconds
But let’s a take a quick step back. Actually ten years back. That’s when Shinya Yamanaka discovered that the insertion of just four genes – let’s call them the Yamanaka genes – into adult skin cells in vitro can wipe their identities clean allowing them to be specialized into virtually any cell type. While this ground breaking work led to a Nobel Prize, the efficiency of the method was very low. Research in the past couple of years has shown in vivo reprogramming is also possible but also at a low efficiency.

So what’s behind the low efficiency? Some culprits include tumor suppressor proteins (which act like kill switches in cells to prevent them from turning into cancer) like p53 and INK4. Blocking the activity of either protein increases the efficiency of in vitro reprogramming. But a funny thing happened in the current study when the researchers did the same thing in vivo. They injected mouse skin cells with the Yamanaka reprogramming genes into mice lacking the p53 gene or the INK4 gene or into control mice with both genes intact. Compared to the control mice, in vivo reprogramming efficiency was higher in the mice missing the p53 gene, as you’d expect based on the in vitro results described above. But in mice without the INK4 gene, the efficiency was actually lower than the control. That’s the exact opposite of the in vitro case in which blocking INK4 increases reprogramming efficiency.

Who knew? Cell slow down stimulates the iPS process in surrounding cells
To investigate this baffling result, the team focused on the fact that INK4 plays a role in cell senescence. When cells get old or damaged they become senescent; that is, they stop dividing and release proteins that cause inflammation. Now, it turns out that in vivo, the Yamanaka genes not only drive reprogramming but they also lead to a lot of damage to surrounding cells causing them to senesce.

And herein lies the answer. The in vivo reprogramming efficiency appears to depend on surrounding cells becoming senescent. Cells in the mice lacking INK4 don’t senesce, and the resulting reprogramming efficiency is low. But in mice lacking p53, the team observed lots of senescent cells along with increased reprogramming efficiency.

By studying the various inflammation-causing proteins that senescent cells release, the team zeroed in on a protein called IL-6 as the connection between reprogramming and senescence. When IL-6 was blocked, in vivo reprogramming efficiency dropped. The team also mimicked these results in vitro. When damaged cells were present while reprogramming cells in the same petri dish, efficiency increased. And when IL-6 was removed from the nutrients the cells were grown in, in vitro reprogramming efficiency decreased.

nov28_2016_science_cellreprogramming4207151976

IL-6 released from damaged cells boosts reprogramming in surrounding cells with the potential for regenerative repair. (Image: Mosteiro et al. Science. 2016 Nov 25;354(6315))

Please note
It’s important to keep in mind that the end goal here is not to find ways to optimize the use of the Yamanaka genes for in vivo reprogramming efficiency in a clinical setting in people. Doing so would carry the dangerous risk of causing cancer. Instead, these results have revealed that senescent cells, through the action of IL-6, appear to stimulate the regeneration and repair of damaged or injured organs. A CNIO press release described how the scientists plan to apply these new insights:

“Having identified the essential role of IL6, … the team [is] now working on various pharmacological approaches to enhance the reprogramming efficiency, which could help to improve the regeneration of damaged tissue even in the absence of the Yamanaka genes. Improving the repairing capacity of tissues could have obvious implications for regenerative medicine, including the treatment of multiple pathologies and degenerative processes associated with ageing.”

Double dose of good news: scientists use drugs to turn skin into heart and brain cells

Today the stem cell field got a double dose of good news. Two CIRM-funded studies from the Gladstone Institutes reported successfully reprogramming human skin into heart cells and brain cells in a dish using different cocktails of small molecule drugs.

Gladstone Investigators Yadong Huang, Sheng Ding, and Deepak Srivastava.

Gladstone Investigators Yadong Huang, Sheng Ding, and Deepak Srivastava.

The work was led by Dr. Sheng Ding, a Senior Investigator at the Gladstone Institutes. Ding is a rock star in the stem cell reprogramming field and has published numerous papers that convincingly show that small molecules are an attractive alternative to genetic reprogramming approaches.

The ability to generate heart and brain tissue from skin without using added genetic components is an achievement that the field of regenerative medicine has been waiting for. These findings point to a future where drugs can be used to direct the body’s existing cells to regenerate damaged or lost tissue.

Comes from the heart

In the first study, which was published in the journal Science, scientists used a cocktail of nine small molecule drugs to directly reprogram human skin cells into beating heart cells called cardiomyocytes.

The team initially screened 89 small molecule drugs that were known to be involved in reprogramming adult cells back to a pluripotent stem cell state. Skin cells were treated with these drugs for six days and then treated with another set of factors that promote heart muscle development. Using this method, they identified a combination of nine compounds (9C) that generated clusters of beating heart cells from skin after 30 days. Upon further investigation, they found that 9C treatment opened up the chromatin landscape (the complex of DNA and protein that make up chromosomes) surrounding genes important for heart development, allowing for the activation of important heart signaling pathways.

Chemically reprogrammed heart cell from skin. (Gladstone)

Chemically reprogrammed heart cell from skin. (Gladstone)

Unlike previous studies whose attempts to generate cardiomyocytes from skin yielded very few heart muscle cells that could beat spontaneously (~0.1%), Ding and his team found that more than 97% of chemically induced cardiomyocytes were able to get their beat on. Furthermore, when they transplanted 9C-treated skin cells into mice that had damaged heart tissue from a heart attack, the cells developed into healthy-looking heart muscle in the damaged area of the heart.

In a Gladstone press release, the director of cardiovascular and stem cell research at the Gladstone, Dr. Deepak Srivastava, explained that the next step in this study is to use small molecules to generate new heart muscle tissue from existing cells in the body:

“The ultimate goal in treating heart failure is a robust, reliable way for the heart to create new muscle cells. Reprogramming a patient’s own cells could provide the safest and most efficient way to regenerate dying or diseased heart muscle.”

Regenerating brain tissue

In the second study published today in the journal Cell Stem Cell, Ding and colleagues used a similar but different combination of nine small molecules (M9) to reprogram mouse skin cells into cells that closely resembled brain stem cells. These induced brain stem cell-like cells were able to generate the three main cell types of the brain – neurons, astrocytes, and oligodendrocytes – both in a dish and when transplanted into the brains of mice.

Chemically induced brain stem cells. (Image courtesy of Gladstone Institute)

Chemically induced brain stem cells. (Image courtesy of Gladstone Institute)

They found that the M9 treatment was effective at turning skin into brain stem cells because it activated neural developmental genes in the skin cells including a key gene required for generating brain stem cells called Sox2.

Co-author and senior investigator Dr. Yadong Huang commented on the potential applications of chemically induced brain stem cells:

“With their improved safety, these neural stem cells could one day be used for cell replacement therapy in neurodegenerative diseases like Parkinson’s disease and Alzheimer’s disease. In the future, we could even imagine treating patients with a drug cocktail that acts on the brain or spinal cord, rejuvenating cells in the brain in real time.”

Reprogramming at the site of injury

Both studies suggest that chemical based approaches to reprogramming have significant advantages over other more invasive methods. Chemical compounds can be easily generated and applied to cells in a controlled manner. They also can interact with the proteins in a cell and activate developmental signaling programs for specific tissues (such as the heart or brain) wit less risk of inserting cancer-causing mutations into the genome.

Ultimately, these studies suggest that a patient’s own cells could be reprogrammed inside their body to generate new healthy tissue that would combat disease and injury.

Sheng Ding commented:

“This method brings us closer to being able to generate new cells at the site of injury in patients. Our hope is to one day treat diseases like heart failure or Parkinson’s disease with drugs that help the heart and brain regenerate damaged areas from their own existing tissue cells. This process is much closer to the natural regeneration that happens in animals like newts and salamanders, which has long fascinated us.”


Disclaimer: I did my PhD at the Gladstone and my former boss is a co-author on one of the studies.

MIT Scientists Recreate Malaria in a Dish to Test Promising Drug Candidates

At the beginning, it feels like the flu: aches, pains and vomiting. But then you begin to experience severe cold and shivering, followed by fever and sweating—a cycle, known as tertian fever, that repeats itself every two days. And that’s when you know: you’ve contracted malaria.

Malaria is caused by Plasmodium parasites and spread to people through the bites of infected mosquitoes

Malaria is caused by Plasmodium parasites and spread to people through the bites of infected mosquitoes

But you wouldn’t be alone. According to the World Health Organization, nearly 200 million people, mostly in Africa, contracted the disease in 2013. Of those, nearly half a million—mainly children—died. There is no cure for malaria, and the parasites that cause the disease are quickly developing resistance to treatments. This is a global public health crisis, and experts agree that in order to halt its spread, they must begin thinking outside the box.

Enter Sangeeta Bhatia, renowned biomedical engineer from the Massachusetts Institute of Technology (MIT)—who, along with her team, has devised a quick and easy way to test out life-saving drug candidates that could give doctors and aid workers on the front lines fresh ammunition.

One of the key hurdles facing scientists has been the nature of the disease’s progression itself. Caused by parasites transmitted via infected mosquitos, the disease first takes hold in the liver. It is only after a few weeks that it enters the blood stream, causing symptoms. By then, the disease is so entrenched within the patient that complete eradication is extremely difficult. Even if the patient recovers, he or she will likely suffer relapses weeks, months or even years later.

The trick, therefore, is to catch the disease before it enters the blood stream. To that effect, several promising drugs have been put forth, and scientists are eager to test them out on liver tissue infected with malaria. Except that they can’t: liver tissue donors are few and far between, and lack the genetic diversity needed for large-scale testing.

Liver-stage malarial infection in iPSC-derived liver cells, eight days after infection. [Credit Ng et al.]

Liver-stage malarial infection in iPSC-derived liver cells, eight days after infection. [Credit Ng et al.]

So Bhatia and her team developed a new solution: they’d make the cells themselves. Reporting in today’s issue of Stem Cell Reports, the team describes how they transformed human skin cells into liver cells, by way of induced pluripotent stem cell (iPS cell) technology. Then, by infecting these cells with the malaria parasite, they could test a variety of drug candidates to see which worked best. As Bhatia explained:

“Our platform can be used for testing candidate drugs that act against the parasite in the early liver stages, before it causes disease in the blood and spreads back to the mosquito vector. This is especially important given the increasing occurrence of drug-resistant strains of malaria in the field.”

Bhatia has long been known for finding innovative solutions to longstanding issues in science and medicine. Just last year, she was awarded the prestigious Lemelson-MIT Prize in part for her invention of a paper-based urine test for prostate cancer.

In this study, the researchers bombarded malaria-infected liver cells with two drugs, called atovaguone and primaquine, each developed to treat the disease specifically at the liver stage.

The results, though preliminary, are promising: the cells responded well to both drugs, underscoring the value of this approach to testing drugs—an approach that many call “disease in a dish.”

The potential utility of “disease in a dish” studies cannot be understated, as it gives researchers the ability to screen drugs on cells from individuals of varying genetic backgrounds, and discover which drug, or drugs, works best for each group.

Shengyong Ng, a postdoctoral researcher in Bhatia’s lab, spoke of what this study could mean for disease research:

“The use of iPSC-derived liver cells to model liver-stage malaria in a dish opens the door to study the influence of host genetics on antimalarial drug efficacy, and lays the foundation for their use in antimalarial drug discovery.”

Find out more about how scientists use stem cells to model disease in a dish in our video series, Stem Cells In Your Face.

Stem Cell Stories that Caught our Eye: Skin Cells to Brain Cells in One Fell Swoop, #WeAreResearch Goes Viral, and Genes Helps Stem Cells Fight Disease

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.

Building a Better Brain Cell. Thanks to advances in stem cell biology, scientists have found ways to turn adult cells, such as skin cells, back into cells that closely resemble embryonic stem cells. They can then coax them into becoming virtually any cell in the body.

But scientists have more recently begun to devise ways to change cells from one type into another without first having to go back to a stem cell-like state. And now, a team from Washington University in St. Louis has done exactly that.

As reported this week in New Scientist, researcher Andrew Yoo and his team used microRNAs—a type of ‘signaling molecule’—to reprogram adult human skin cells into medium spiny neurons(MSNs), the type of brain cell involved in the deadly neurodegenerative condition, Huntington’s disease.

“Within four weeks the skin cells had changed into MSNs. When put into the brains of mice, the cells survived for at least six months and made connections with the native tissue,” explained New Scientist’s Clare Wilson.

This process, called ‘transdifferentiation,’ has the potential to serve as a faster, potentially safer alternative to creating stem cells.

#WeAreResearch Puts a Face on Science. The latest research breakthroughs often focus on the science itself, and deservedly so. But exactly who performed that research, the close-knit team who spent many hours at the lab bench and together worked to solve a key scientific problem, can sometimes get lost in the shuffle.

#WeAreResearch submission from The Thomson Lab at the University of California, San Francisco. This lab uses optogenetics, and RNAseq to probe cell fate decisions.

#WeAreResearch submission from The Thomson Lab at the University of California, San Francisco. This lab uses optogenetics, and RNAseq to probe cell fate decisions.

Enter #WeAreResearch, a new campaign led by the American Society for Cell Biology (ASCB) that seeks to show off science’s more ‘human side.’

Many California-based stem cell teams have participated—including CIRM grantee Larry Goldstein and his lab!

Check out the entire collection of submissions and, if you’re a member of a lab, submit your own. Prizes await the best submissions—so now’s your chance to get creative.

New Genes Help Stem Cells Fight Infection. Finally, UCLA scientists have discovered how stem cells ‘team up’ with a newly discovered set of genes in order to stave off infection.

Reporting in the latest issue of the journal Current Biology, and summarized in a UCLA news release, Julian Martinez-Agosto and his team describe how two genes—adorably named Yorkie and Scalloped—set in motion a series of events, a molecular Rube Goldberg device, that transforms stem cells into a type of immune system cell.

Importantly, the team found that without these genes, the wrong kind of cell gets made—meaning that these genes play a central role in the body’s healthy immune response.

Mapping out the complex signaling patterns that exist between genes and cells is crucial as researchers try and find ways to, in this case, improve the body’s immune response by manipulating them.

Harder, Better, Faster, Stronger: Scientists Work to Create Improved Immune System One Cell at a Time

The human immune system is the body’s best defense against invaders. But even our hardy immune systems can sometimes be outpaced by particularly dangerous bacteria, viruses or other pathogens, or even by cancer.

Salk Institute scientists have developed a new cellular reprogramming technique that could one day boost a weakened immune system.

Salk Institute scientists have developed a new cellular reprogramming technique that could one day boost a weakened immune system.

But what if we could give our immune system a boost when it needs it most? Last week scientists at the Salk Institute for Biological Sciences devised a new method of doing just that.

Reporting in the latest issue of the journal Stem Cells, Dr. Juan Carlos Izpisua Belmonte and his team announce a new method of creating—and then transplanting—white blood cells into laboratory mice. This new and improved method could have significant ramifications for how doctors attack the most relentless disease.

The authors achieved this transformation through the reprogramming of skin cells into white blood cells. This process builds on induced pluripotent stem cell, or iPS cell, technology, in which the introduction of a set of genes can effectively turn one cell type into another.

This Nobel prize-winning approach, while revolutionary, is still a many months’ long process. In this study, the Salk team found a way to shorten the cellular ‘reprogramming’ process from several months to just a few weeks.

“The process is quick and safe in mice,” said Izpisua Belmonte in a news release. “It circumvents long-standing obstacles that have plagued the reprogramming of human cells for therapeutic and regenerative purposes.”

Traditional reprogramming methods change one cell type, such as a skin cell, into a different cell type by first taking them back into a stem cell-like, or ‘pluripotent’ state. But here, the research team didn’t take the cells all the way back to pluripotency. Instead, they simply wiped the cell’s memory—and gave it a new one. As first author Dr. Ignacio Sancho-Martinez explained:

“We tell skin cells to forget what they are and become what we tell them to be—in this case, white blood cells. Only two biological molecules are needed to induce such cellular memory loss and to direct a new cell fate.”

This technique, which they dubbed ‘indirect lineage conversion,’ uses the molecule SOX2 to wipe the skin cell’s memory. They then use another molecule called miRNA 125b to reprogram the cell into a white blood cell.

These newly generated cells appear to engraft far better than cells derived from traditional iPS cell technology, opening the door to therapies that more effectively introduce these immune cells into the human body. As Sanchi-Martinez so eloquently stated:

“It is fair to say that the promise of stem cell transplantation is now closer to realization.”