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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Stories that caught our eye: stem cell transplants help put MS in remission; unlocking the cause of autism; and a day to discover what stem cells are all about

multiple-sclerosis

Motor neurons

Stem cell transplants help put MS in remission: A combination of high dose immunosuppressive therapy and transplant of a person’s own blood stem cells seems to be a powerful tool in helping people with relapsing-remitting multiple sclerosis (RRMS) go into sustained remission.

Multiple sclerosis (MS) is an autoimmune disorder where the body’s own immune system attacks the brain and spinal cord, causing a wide variety of symptoms including overwhelming fatigue, blurred vision and mobility problems. RRMS is the most common form of MS, affecting up to 85 percent of people, and is characterized by attacks followed by periods of remission.

The HALT-MS trial, which was sponsored by the National Institute of Allergy and Infectious Diseases (NIAID), took the patient’s own blood stem cells, gave the individual chemotherapy to deplete their immune system, then returned the blood stem cells to the patient. The stem cells created a new blood supply and seemed to help repair the immune system.

Five years after the treatment, most of the patients were still in remission, despite not taking any medications for MS. Some people even recovered some mobility or other capabilities that they had lost due to the disease.

In a news release, Dr. Anthony Fauci, Director of NIAID, said anything that holds the disease at bay and helps people avoid taking medications is important:

“These extended findings suggest that one-time treatment with HDIT/HCT may be substantially more effective than long-term treatment with the best available medications for people with a certain type of MS. These encouraging results support the development of a large, randomized trial to directly compare HDIT/HCT to standard of care for this often-debilitating disease.”

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Scripps Research Institute

Using stem cells to model brain development disorders. (Karen Ring) CIRM-funded scientists from the Scripps Research Institute are interested in understanding how the brain develops and what goes wrong to cause intellectual disabilities like Fragile X syndrome, a genetic disease that is a common cause of autism spectrum disorder.

Because studying developmental disorders in humans is very difficult, the Scripps team turned to stem cell models for answers. This week, in the journal Brain, they published a breakthrough in our understanding of the early stages of brain development. They took induced pluripotent stem cells (iPSCs), made from cells from Fragile X syndrome patients, and turned these cells into brain cells called neurons in a cell culture dish.

They noticed an obvious difference between Fragile X patient iPSCs and healthy iPSCs: the patient stem cells took longer to develop into neurons, a result that suggests a similar delay in fetal brain development. The neurons from Fragile X patients also had difficulty forming synaptic connections, which are bridges that allow for information to pass from one neuron to another.

Scripps Research professor Jeanne Loring said that their findings could help to identify new drug therapies to treat Fragile X syndrome. She explained in a press release;

“We’re the first to see that these changes happen very early in brain development. This may be the only way we’ll be able to identify possible drug treatments to minimize the effects of the disorder.”

Looking ahead, Loring and her team will apply their stem cell model to other developmental diseases. She said, “Now we have the tools to ask the questions to advance people’s health.”

A Day to Discover What Stem Cells Are All about.  (Karen Ring) Everyone is familiar with the word stem cells, but do they really know what these cells are and what they are capable of? Scientists are finding creative ways to educate the public and students about the power of stem cells and stem cell research. A great example is the University of Southern California (USC), which is hosting a Stem Cell Day of Discovery to educate middle and high school students and their families about stem cell research.

The event is this Saturday at the USC Health Sciences Campus and will feature science talks, lab tours, hands-on experiments, stem cell lab video games, and a resource fair. It’s a wonderful opportunity for families to engage in science and also to expose young students to science in a fun and engaging way.

Interest in Stem Cell Day has been so high that the event has already sold out. But don’t worry, there will be another stem cell day next year. And for those of you who don’t live in Southern California, mark your calendars for the 2017 Stem Cell Awareness Day on Wednesday, October 11th. There will be stem cell education events all over California and in other parts of the country during that week in honor of this important day.

 

 

A TWIST in mesenchymal stem cell trials: protein predicts therapy’s potential

Mesenchymal stem cells are adult stem cells with the potential to specialize into a somewhat limited number of cell types – those responsible for making fat, bone and cartilage. But MSCs are also known for their anti-inflammatory properties which are carried out via the release of protein factors.  This ability to dampen the immune system makes the MSC an extremely attractive source material for cell therapies. In fact, there are over 500  mesenchymal stem cell-based clinical trials testing treatments for diseases that target a wide range of tissues including spinal cord injury, diabetes, multiple sclerosis, respiratory disorders and graft versus host disease, just to name a few.

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Human mesenchymal stem cells grown in a single layer on the bottom of a flask; 4x magnification Image source: EuroStemCell

 MSCs and the Variability Problem
While some MSC-based human trials have had promising results in patients, other studies haven’t been as successful. A key culprit of these mixed results is the lack of standardization on what exactly is a MSC. It’s well documented that preparations of MSC vary significantly from one patient to the next. Even the composition of MSCs from one patient is far from a pure population of cells. And few of the cell surface markers used to define MSCs provide a measure of the cells’ function. This is a real problem for demonstrating the effectiveness and the marketability of MSC-based cell therapies which rely on the delivery of cell product with a consistent, well-defined composition and functional activity.

Help is now on the way based on research reported this week in EBioMedicine by a research team led by Professor Donald Phinney at the Florida campus of The Scripps Research Institute. In the study, the team found that the amount of TWIST1, a protein that regulates gene activity, in a given batch of MSCs could reliably predict the therapeutic effectiveness of those cells.

Meet TWIST1: predictor of a MSC therapy’s potential
They set their sights on TWIST1 because previous research described its important role in driving a MSC fate during human development. The team examined the natural variability of TWIST1 levels in human MSCs from several donors. They showed that lower levels of TWIST1 correlated to MSCs with stronger anti-inflammatory properties. Higher levels of TWIST1, on the other hand, were consistent with MSCs that induced angiogenesis, or blood vessel growth, another known ability of this versatile cell type. In another set of experiments, TWIST1 production was silenced using genetic tools. As predicted by the earlier results, these MSCs showed increased anti-inflammatory properties.

Move over Ritcher, Say Hello to the CLIP Scale

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The Clinical Indication Prediction (CLIP) scale. Image: Boregowda et al. EBioMedicine, Volume 4 , 62-73

Putting this data together, the team devised a scale they call Clinical Indication Prediction, or CLIP for short. The scale gives a clinical researcher an indication of the therapeutic potential of a given batch of donor MSCs based on the TWIST1 protein levels. This information could have a major impact on a clinical trial’s fate. Depending on the goal of a MSC-based cell therapy, a clinical team could set themselves up for failure before the trial even gets underway if they don’t take TWIST1 levels into account. First author Siddaraju V. Boregowda explains this scenario in a press release:

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Siddaraju V. Boregowda

“There are a number of clinical trials testing mesenchymal stem cells to treat arthritis. Since angiogenesis is a key part of the disease process, stem cells with high levels of TWIST1 (indicating they are more angiogenic) would not be beneficial. These cells might be helpful instead for indications such as peripheral vascular disease where new vascularization is beneficial. The proposed CLIP scale accurately predicts these indications and contra-indications.”

We’ll be keeping our eye on this exciting discovery to see if CLIP becomes an integral step in developing MSC-based cell therapies. If it pans out, the CLIP scale could help accelerate the development of new therapies by providing scientists with more clarity and confidence around classifying the identity of a MSC cell product. Stay tuned!

Cell survival strategy gives mesenchymal stem cells their “paramedic” properties

Electron micrograph of a human mesenchymal stem cells (Credit: Robert M. Hunt)

Electron micrograph of a human mesenchymal stem cells (Image credit: Robert M. Hunt)

A cell for all therapies
Type “mesenchymal stem cells” into the federal online database of registered clinical trials, and you’ll get a sprawling list of 527 trials testing treatments for diabetes, multiple sclerosis as well as diseases of the kidney, lung, and heart, to name just a few. Mesenchymal stem cells (MSCs) have the capacity to specialize into bone, cartilage, muscle and fat cells but their popularity as a therapeutic agent mostly comes from their ability to reduce inflammation and to help repair tissues.

MSCs may be great tools for scientists to fight disease, but what is it about their natural function that make MSCs – as UC Davis researcher Jan Nolta likes to calls them – the body’s “paramedics”? A fascinating study reported yesterday in Nature Communications by scientists at the Florida campus of The Scripps Research Institute (TSRI) and the University of Pittsburgh suggest that it’s a trait the cells gain as a result of their complex cell survival mechanisms.

The TSRI team came to this conclusion by studying how MSCs respond to oxygen-related stress. MSCs reside in the bone marrow where they help maintain and regulate blood stem cells. The bone marrow is naturally a hypoxic, or low oxygen, environment. Growing MSCs in the lab at oxygen levels found in the air we breathe are much higher than what is found in the marrow. This creates oxidative stress in which the excess oxygen leads to unwanted chemical reactions which disrupt a cell’s molecules.

One cell’s trash is another’s treasure
One result of this oxidative stress is damage to the MSCs’ mitochondria, structures responsible for generating the energy needs of a cell. The team found that MSCs package the faulty mitochondria into sacs, or vesicles, which travel to the cell surface to be dumped out of the cell. At this point, another resident of the bone marrow comes into the picture: the macrophage. Previous research has shown that macrophages and MSCs work closely together to maintain the health of the blood stem cells in the bone marrow.

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White arrow shows vesicles (red) carrying mitochondra (green) to the surface of the MSC  and being ingested by a macrophage (round shape in lower half) – (From Fig 2 Nat Commun. 2015 Oct 7;6:8472)

In a high oxygen stress environment, the team observed that MSCs can recruit macrophages to engulf the damaged mitochondria-containing vesicles and repurpose them for their own use. In fact, the researchers measured improved energy production in the macrophages after ingesting the MSCs’ mitochondria. Blocking the transfer of the damaged mitochondria from MSCs to macrophages caused the MSCs to die, confirming that this off-loading of mitochondria to macrophages is critical for MSC survival.

Evolving tricks for cell survival
Macrophages (macro=big; phages=eaters), key players of the immune system and the inflammation response, also rid the body of invading bacteria or damaged cells by devouring them. To avoid being swallowed up by the macrophage while donating its mitochondria, the stressed MSCs have another trick up their sleeve. The research team identified the release of other vesicles from the MSCs that contain molecules called microRNAs which stimulate anti-inflammatory properties in the macrophages. This prevented the macrophages from attacking and eating the MSCs.

And there you have it: as a result of relying on macrophages to survive stressful environments, MSCs appear to have evolved anti-inflammatory activities that turn out to be a handy tool for numerous ongoing and future cell therapy trials.

In a TSRI press release picked up by Newswise, professor Donald Phinney co-leader of study points out the groundbreaking aspect of the study:

Donald G. Phinney

Donald Phinney (photo: TSRI)

“This is the first time anyone has shown how mesenchymal stem cells provide for their own survival by recruiting and then suppressing normal macrophage activity.”

 

 

Goodnight, Stem Cells: How Well Rested Cells Keep Us Healthy

Plenty of studies show that a lack of sleep is nothing but bad news and can contribute to a whole host of health problems like heart disease, poor memory, high blood pressure and obesity.

HSCs_Sleeping_graphic100x100

Even stem cells need rest to stay healthy

In a sense, the same holds true for the stem cells in our body. In response to injury, adult stem cells go to work by dividing and specializing into the cells needed to heal specific tissues and organs. But they also need to rest for long-lasting health. Each cell division carries a risk of introducing DNA mutations—and with it, a risk for cancer. Too much cell division can also deplete the stem cell supply, crippling the healing process. So it’s just as important for the stem cells to assume an inactive, or quiescent, state to maintain their ability to mend the body. Blood stem cells for instance are mostly quiescent and only divide about every two months to renew their reserves.

Even though the importance of this balance is well documented, exactly how it’s achieved is not well understood; that is, until now. Earlier this week, a CIRM-funded research team from The Scripps Research Institute (TSRI) reported on the identification of an enzyme that’s key in controlling the work-rest balance in blood stem cells, also called hematopoietic stem cells (HSCs). Their study, published in the journal Blood, could point the way to drugs that treat anemias, blood cancers, and other blood disorders.

Previous studies in other cell types suggested that this key enzyme, called ItpkB, might play a role in promoting a rested state in HSCs. Senior author Karsten Sauer explained their reasoning for focusing on the enzyme in a press release:

“What made ItpkB an attractive protein to study is that it can dampen activating signaling in other cells. We hypothesized that ItpkB might do the same in HSCs to keep them at rest. Moreover, ItpkB is an enzyme whose function can be controlled by small molecules. This might facilitate drug development if our hypothesis were true.”

Senior author Karsten Sauer is an associate professor at The Scripps Research Institute.

Senior author Karsten Sauer is an associate professor at The Scripps Research Institute.

To test their hypothesis, the team studied HSCs in mice that completely lacked ItpkB. Sure enough, without ItpkB the HSCs got stuck in the “on” position and continually multiplied until the supply of HSCs stores in the bone marrow were exhausted. Without these stem cells, the mice could no longer produce red blood cells, which deliver oxygen to the body or white blood cells, which fight off infection. As a result the animals died due to severe anemia and bone marrow failure. Sauer used a great analogy to describe the result:

“It’s like a car—you need to hit the gas pedal to get some activity, but if you hit it too hard, you can crash into a wall. ItpkB is that spring that prevents you from pushing the pedal all the way through.”

With this new understanding of how balancing stem cell activation and deactivation works, Sauer and his team have their sights set on human therapies:

“If we can show that ItpkB also keeps human HSCs healthy, this could open avenues to target ItpkB to improve HSC function in bone marrow failure syndromes and immunodeficiencies or to increase the success rates of HSC transplantation therapies for leukemias and lymphomas.”

CIRM-Funded Scripps Team Replicates Pain in a Lab Dish; Seeks New Treatments for Chronic Sufferers

Pain hurts but it also protects. Thanks to nerve cells called sensory neurons, which weave their nerve fibers throughout our skin and other tissues, we are alerted to dangerous events like touching a hot plate or even to the sense of having a full bladder.

However, trauma such as a spinal cord injury or diseases like HIV and diabetes can damage sensory neurons and cause chronic pain that debilitates rather than protects those affected. Sadly, conventional pain treatments are usually not effective for the stinging, burning, tingling and numbness associated with this type of pain. Clearly, new innovations are needed.

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These induced sensory neurons could be useful in the testing of potential new therapies for pain, itch and related conditions. Credit: Baldwin Lab, The Scripps Research Institute

Last week, a CIRM-funded research team from The Scripps Research Institute, reported in Nature Neuroscience that they developed a technique, which induces human skin cells to transform into sensory neurons in a petri dish. Up until now, the field mostly relied on mouse studies due to the difficulty of collecting and growing human sensory neurons in the lab. This may explain the lack of success in clinical trials for treating chronic pain. As co-lead author Joel Blanchard, a PhD candidate in Kristin Baldwin’s laboratory, stated in the institute’s press release:

“Mouse models don’t represent the full diversity of the human response. [With these human sensory neurons] we can start to understand how individuals respond uniquely to pain, cold, itch and so on.”

Kevin Eade, research associate, and Joel Blanchard, graduate student, co-lead authors of the report  Credit: Cindy Brauer, The Scripps Research Institute

Kevin Eade, research associate, and Joel Blanchard, graduate student, co-lead authors of the report. Credit: Cindy Brauer, The Scripps Research Institute

To generate the nerve cells, the Baldwin research team inserted, into human skin cells, a combination of genes known to produce proteins that are key controllers of sensory neuron function. The resulting cells had the appearance of sensory neurons and responded appropriately when exposed to heat in the form of the active ingredient in chili peppers as well as activating a cold response when exposed to menthol. Adding more confidence to these results, an independent research team from the Harvard Stem Cell Institute reported in the same Nature Neuroscience   issue and in a press release that they too had successfully generated human sensory neurons from skin cells.

This direct reprogramming of one cell type directly into another is a variant of the induced pluripotent stem cell (iPS) technique in which a cell, often skin, is first reprogrammed into an embryonic stem cell-like state and then coaxed to form into virtually any cell type of the body.

Now that the Baldwin lab has nailed down the recipe for making human sensory neurons, they now can seek out treatments to bring relief to chronic pain sufferers. Dr. Baldwin looks forward to this future work:

Kristin Baldwin, Associate Professor Department of Molecular and Cellular Neuroscience. Credit: The Scripps Research Institute

Kristin Baldwin
Associate Professor
Credit: The Scripps Research Institute

“This method is rapid, robust and scalable. Therefore we hope that these induced sensory neurons will allow our group and others to identify new compounds that block pain and itch and to better understand and treat neurodegenerative disease and spinal cord injury.”

Watch the short video below to hear from a pioneer of direct reprogramming of nerve cells, CIRM grantee Marius Wernig of Stanford University: