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

CRISPR

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

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

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

Reviewing the rules

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

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

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

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

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

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

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

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

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

Time to ask tough questions

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

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

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

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

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

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

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

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

Stay tuned, more to come!

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

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

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

How you derive embryonic stem cells matters

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

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

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

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

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

Culturing methods erase stem cell memory

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

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

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

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

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

Amandar Clark, UCLA

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

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

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

How you derive embryonic stem cells matters

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

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


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

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

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

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

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

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

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

 

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

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

 

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

compressingc

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

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

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

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

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

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

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

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

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

200mg_ibuprofen_tablets

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

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

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

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

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

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

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

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

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

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

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

stemgellbandaid.jpg

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

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

A Tale of Two Stem Cell Treatments for Growing New Bones

Got Milk?

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

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

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

Protein Combo Boosts Bone Growth

Osteoporosis. (Image source)

Osteoporosis. (Image source)

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

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

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

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

Kang Ting, UCLA

Kang Ting, UCLA

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

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

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

Stem cells that could fix deformed skulls

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

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

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

1651177064_WeiHsu-stem cell photo_4487_275x200

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

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

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

Two different studies, one common goal

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

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

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


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

Buckle up

Buckle up, we’re about to Accelerate

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

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

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

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

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

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

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

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

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

 

 

 

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

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

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

Penetrating the Impenetrable

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

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

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

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

A Virus that Makes Your Brain Glow Green

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

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

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

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

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

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

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

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

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

Sergiu Pasca

Sergiu Pasca

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

An easier way to deliver genes across the BBB

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

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

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


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

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

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

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

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

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

 

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

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

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

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

 

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

a6353-spinalcord

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

 

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

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

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

 

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

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

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

3D printed human ear.

3D printed human ear.

Mini-stomachs.

Mini-stomachs.

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

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

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

Med-Bays.

Med-Bays.

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

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

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

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

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

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

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

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

Vinit Mahajan

Vinit Mahajan

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

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

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


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New drug kicks the cancer stem cell addiction

Did you know that cancer stem cells have an addiction problem? This might sound bizarre, but the science checks out.

Cancer stem cells are found in many different types of cancer tumors. They have the uncanny ability to survive even the most aggressive forms of treatment. After weathering the storm, cancer stem cells are able to divide and repopulate an entire tumor and even take road trips to create tumors in other areas of the body.

How cancer stem cells are able to survive and thrive is a question that is being actively pursued by scientists who aim to develop new strategies that target these cells.

Cancer stem cells have a Wnt addiction

To understand why a cancer stem cell is so good at staying alive and creating new tumors, you need to get down to the protein signaling level, which is basically a cascade of protein interactions that begin at the cell surface and instruct certain activities inside the cell. During embryonic development, one of the signaling pathways that’s activated is the Wnt pathway. It’s responsible for keeping embryonic stem cells in a pluripotent state where they maintain the ability to become any cell type.

As embryonic stem cells mature into adult cells, Wnt signaling plays different roles. It helps stem cells differentiate or change into cells of various tissues and helps maintain the health and integrity of those tissues. Because Wnt signaling has varying functions depending on the developmental stage of the cells, it’s important for cells to properly regulate this pathway.

It turns out that cancer stem cells don’t do this. Typically cells need to receive certain biochemical signals to activate the Wnt pathway, but cancer stem cells acquire genetic mutations and evolve such that this pathway is constantly activated. They ramp up their Wnt signaling and never turn it off. This “Wnt addiction” allows them to stay alive and flourish in a cancerous stem cell state.

Kicking the Wnt Addiction

A team at the Max Delbruck Center (MDC) in Germany decided to kick this Wnt addiction and make cancer stem cells go cold turkey. They published their results in the journal Cancer Research this week.

Their strategy involved targeting proteins called transcription factors, the activators of genes, that are turned on during aberrant Wnt signaling in cancer stem cells. The transcription factor they focused on is called TCF4. In normal cells, biochemical signals are required to activate the Wnt cascade and a protein called beta-catenin, which transmits signals to transcription factors like TCF4 that then turn on genes. In cancer stem cells, this signal isn’t required because the Wnt pathway is permanently switched on leaving TCF4 free to activate genes that promote tumor cell survival and growth.

The researchers thought that if they could break up the partnership between beta-catenin and TCF4, that they might be able to block Wnt signaling and kill the life-line of the cancer stem cells. They screened a library of drugs and identified a small molecule called LF3 that was able to block the interaction between beta-catenin and TCF4.

A new drug kills that cancer stem cells. The image on the left shows beta catenin (red) in cell nuclei indicating that these are cancer stem cells. The image on the right shows that the new substance sucessfully removed beta catenin from the nuclei. Picture by Liang Fang for the MDC

Cancer stem cells express beta-catenin shown in red on the left. On the right, drug treatment blocks Wnt signaling and removes beta-catenin from the cancer stem cells. (Image: Liang Fang for the MDC)

The scientists tested the LF3 molecule in mice with tumors derived from human colon cancer stem cells. Senior author on the study, Walter Birchmeier, explained in an MDC press release:

Walter

Walter Birchmeier

“We observed a strong reduction of tumor growth. What remained of the tumors seemed to be devoid of cancer stem cells – LF3 seemed to be powerfully triggering these cells to differentiate into benign tissue. At the same time, no signaling systems other than Wnt were disturbed. All of these factors make LF3 very promising to further develop as a lead compound, aiming for therapies that target human tumors whose growth and survival depend on Wnt signaling.”

Upon further analysis, they found that LF3 prevented cancer stem cells from dividing into more stem cells and migrating to other tissues. Instead, they differentiated into non-cancerous tissues. Importantly, the drug did not negatively affect the function of healthy cells nearby. This is a logical concern as Wnt signaling is activated in healthy adult tissue, just in a different way than in stem cells.

This study offers a new angle for cancer treatment. Not only does LF3 force cancer stem cells to kick their “Wnt addiction”, it also spares healthy cells and tissues. This drug sounds like a promising option for patients who suffer from aggressive, recurring tumors caused by cancer stem cells.


 

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