Here’s a new gene editing strategy to treat genetic blood disorders

If you’re taking a road trip across the country, you have a starting point and an ending point. How you go from point A to point B could be one of a million different routes, but the ultimate outcome is the same: reaching your final destination.

Yesterday scientists from St. Jude Children’s Research Hospital published exciting findings in the journal Nature Medicine on a new gene editing strategy that could offer a different route for treating genetic blood disorders such as sickle cell disease (SCD) and b-thalassemia.

The scientists used a gene editing tool called CRISPR. Unless you’ve been living under a rock, you’ve heard about CRISPR in the general media as the next, hot technology that could possibly help bring cures for serious diseases.

In simple terms, CRISPR acts as molecular scissors that facilitate cutting and pasting of DNA sequences at specific locations in the genome. For blood diseases like SCD and b-thalassemia, in which blood cells have abnormal hemoglobin, CRISPR gene editing offers ways to turn on and off genes that cause the clinical symptoms of these diseases.

Fetal vs. Adult hemoglobin

Before I get into the meat of this story, let’s take a moment to discuss hemoglobin. What is it? It’s a protein found in red blood cells that transports oxygen from the lungs to the rest of the body. Hemoglobin is made up of different subunits and the composition of these hemoglobin subunits change as newborns develop into adults.

0a448-sicklecellimage

Healthy red blood cell (left), sickle cell (right).

Fetal hemoglobin (HbF) is comprised of a and g subunits while adult hemoglobin (HbA) is typically comprised of a and b subunits. Patients with SCD and b-thalassemia typically have mutations in the b globin gene. In SCD, this causes blood cells to take on an unhealthy, sickle cell shape that can clog vessels and eventually cause premature death. In b-thalassemia, the b-globin gene isn’t synthesized into protein at the proper levels and patients suffer from anemia (low red blood cell count).

One way that scientists are attempting to combat the negative side effects of mutant HbF is to tip the scales towards maintaining expression of the fetal g-globin gene. The idea spawned from individuals with hereditary persistence of fetal hemoglobin (HPFH), a condition where the hemoglobin composition fails to transition from HbF to HbA, leaving high levels of HbF in adult blood. Individuals who have HPFH and are predisposed to SCD or b-thalassemia amazingly don’t have clinical symptoms, suggesting that HbF plays either a protective or therapeutic role.

The current study is taking advantage of this knowledge in their attempt to treat blood disorders. Mitchell Weiss, senior author on the study and chair of the St. Jude Department of Hematology, explained the thought process behind their study:

“It has been known for some time that individuals with genetic mutations that persistently elevate fetal hemoglobin are resistant to the symptoms of sickle cell disease and beta-thalassemia, genetic forms of severe anemia that are common in many regions of the world. We have found a way to use CRISPR gene editing to produce similar benefits.”

CRISPRing blood stem cells for therapeutic purposes

Weiss and colleagues engineered red blood cells to have elevated levels of HbF in hopes of preventing symptoms of SCD. They used CRISPR to create a small deletion in a sequence of DNA, called a promoter, that controls expression of the hemoglobin g subunit 1 (HBG1) gene. The deletion elevates the levels of HbF in blood cells and closely mimics genetic mutations found in HPFH patients.

Weiss further explained the genome editing process in a news release:

Mitchell Weiss

Mitchell Weiss

“Our work has identified a potential DNA target for genome editing-mediated therapy and offers proof-of-principle for a possible approach to treat sickle cell and beta-thalassemia. We have been able to snip that DNA target using CRISPR, remove a short segment in a “control section” of DNA that stimulates gamma-to-beta switching, and join the ends back up to produce sustained elevation of fetal hemoglobin levels in adult red blood cells.”

The scientists genetically modified hematopoietic stem cells and blood progenitor cells from healthy individuals to make sure that their CRISPR gene editing technique was successful. After modifying the stem cells, they matured them into red blood cells in the lab and observed that the levels of HbF increased from 5% to 20%.

Encouraged by these results, they tested the therapeutic potential of their CRISPR strategy on hematopoietic stem cells from three SCD patients. While 25% of unmodified SCD blood stem cells developed red blood cells with a sickle cell shape under low-oxygen conditions (to induce stress), CRISPR edited SCD stem cells generated way fewer sickle cells (~4%) and had a higher level of HbF expression.

Many routes, one destination

The authors concluded that their genome editing technique is successful at switching hemoglobin expression from the adult form back to the fetal form. With further studies and safety testing, this strategy could be one day be developed into a treatment for patients with SCD and b-thalassemia

But the authors were also humble in their findings and admitted that there are many different genome editing strategies or routes for developing therapies for inherited blood diseases.

“Our results represent an additional approach to these existing innovative strategies and compare favorably in terms of the levels of fetal hemoglobin that are produced by our experimental system.”

My personal opinion is the more strategies thrown into the pipeline the better. As things go in science, many of these strategies won’t be successful in reaching the final destination of curing one of these diseases, but with more shots on goal, our chances of developing a treatment that works there are a lot higher.


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Tunable hydrogels guide stem cell differentiation

Differentiating stem cells into mature cells of adult tissue involves many intricate steps to get them to develop into the right cell types. You could compare the process to the careful adjustments you make when tuning a guitar.

In the body, stem cells receive cues from their surrounding environment to mature into specific types of cells. These cues can be biochemical – molecules like lipids, growth factors and metabolites (products of cell metabolism) – or they can be physical – the stiffness of surrounding tissue. But these molecules and structures aren’t always present when scientists attempt to differentiate stem cells outside the body, say in a cell culture dish.

One way researchers are improving the methods for differentiating stem cells outside the body is by using biomaterials such as hydrogels that mimic properties of the structures and molecules found naturally in various stem cell niches that aid in their maturation to adult cell types.

A CIRM-funded study published last week in the journal Chem, has developed “tunable hydrogels” that direct stem cells to differentiate into brain, cartilage and bone cells based on adjustments to the hydrogel’s stiffness and metabolite profile. The work was a collaboration between scientists in New York and in Scotland and one of the co-authors, Bruno Péault, was a CIRM-funded scientist in California during the time of the study.

Hydrogels with different stiffness' direct stem cells to differentiate into different types of tissue. (Chem)

Hydrogels with different stiffness’ direct stem cells to differentiate into different types of tissue. (Chem)

Tuning gels to differentiate stem cells

The scientists started with hydrogels composed of nanofibers that varied in stiffness and observed that perivascular stem cells (from the connective tissue surrounding blood vessels) grown in more flexible gels turned into brain cells and those that were grown in stiffer gels turned into bone cells. The stiffness of these different hydrogels was comparable to that of actual brain and bone tissue, which indicated that stiffness is important for stem cell fate.

But stiffness alone isn’t responsible for directing stem cells into different cell fates – biochemical metabolites are also key to this process. The authors also analyzed the metabolite profiles of the different hydrogels to determine which metabolites were important for stem cell differentiation. They tested different concentrations of over 600 metabolites in the hydrogels during stem cell differentiation and found that certain lipids like lysophosphatidic acid and cholesterol sulfate were essential for differentiation into cartilage and bone tissue respectively. When these specific lipids were added to regular stem cell cultures (without hydrogels), the stem cells differentiated towards cartilage and bone cells. Thus they concluded that both the metabolite profile and the stiffness of hydrogels are important for directing stem cell differentiation.

Interestingly, the authors also showed how metabolites like cholesterol sulfate could influence and activate transcription factors – proteins that also direct stem cell differentiation – which controlled the activation of bone-related genes. This finding suggests a relationship between metabolite expression and transcription factor activity and offers a simpler way to activate transcription factors important for stem cell fate.

Big picture of tunable hydrogels

Looking at the big picture, this study offers a useful strategy to identify molecules that promote formation of specific tissue types from stem cells. These molecules could be potential drug candidates that could aid in regenerating bone and cartilage tissue for patients with osteoporosis or osteoarthritis.

Co-senior author on the study and professor at the University of Glasgow, Matthew Dalby, who was interviewed by Science Magazine elaborated on the importance of their study:

Matthew Dalby

Matthew Dalby

“Our ambition is to simplify drug discovery — by using the cell’s own metabolites as drug candidates. For example, cholesterol sulfate, which our rigid gel revealed as critical to bone cell differentiation, could be a safer solution (e.g., minimal off-target effects) for treating osteoporosis, spinal fusion, and other bone-related conditions. Presently, growth factors are used, but these can lead to unwanted collateral damage, and government agencies in the UK and US have published warnings against their use.”

Rein Ulijn, co-senior author with Dalby and professor at the City University of New York and University of Strathclyde, concluded by emphasizing how the metabolites they identified could be potential drug candidates and would pass regulatory approval if shown to be safe and effective:

Rein Ulijn

Rein Ulijn

“That you can use simple metabolites like cholesterol sulfate, which is readily available, to induce differentiation is in my view very powerful if you think about this as a potential drug candidate. These metabolites are inherently biocompatible, so the hurdles to approval are going to be much lower compared to those associated with completely new chemical entities.”

In the future, both teams plan to further “tune” their hydrogels to mimic more complex tissue environments that incorporate additional properties besides stiffness in hopes of creating more relevant 3D micro-environments to model the stem cell niche.

Stem cell stories that caught our eye: growing muscle, new blood vessels and pacemakers and Tommy John surgery

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.

Better way to grow muscle.  The specialized stem cells responsible for repairing muscle, the satellite cells, have always been difficult to grow in large quantities in the lab. They have a strong natural hankering to mature into muscle. Researchers have not been able to keep them in their stem cell state in the lab and that prevents creating enough of them for effective therapies for diseases like muscular dystrophy.

new muscle Kodaira

New muscle fibers in green grown in mice from satellite stem cells

A team at the National Institute of Neuroscience in Kodaira, Japan, published what seems to be a simple solution to the problem. In a press release from the publisher of the Journal of Neuromuscular Diseases posted by Science Daily they reported that adding just one protein to satellite cells allowed them to grow indefinitely in the lab and expand to the point they could provide a meaningful transplant that resulted in muscle repair in mice.

 “This research enables us to get one step closer to the optimal culture conditions for muscle stem cells,” said Shin’ichi Takeda from the institute.

The protein they used, leukemia inhibitory factor, and its downstream impacts on other genes is now the subject of their ongoing research.

 

Regenerating heart vessels. A CIRM funded team at Sanford Burnham Prebys Medical Discovery Institute (SBP) in San Diego and at Stanford University have shown that repressing a single gene can encourage the formation of new blood vessels in the heart. Creating those new conduits for oxygen after a heart attack could reduce damage to the heart muscle and prevent development of heart failure.

Building new blood vessels requires coordination of several growth factors and clinical trials evaluating individual factors have resulted in failure. The SBP team found that a single gene repressed all those needed factors and blocking it could let them do their job and create new blood vessels.

Mark Mercola

Mark Mercola

“We found that a protein called RBPJ serves as the master controller of genes that regulate blood vessel growth in the adult heart,” said senior author Mark Mercola, a professor at SBP and at Stanford, in an institute press release. “RBPJ acts as a brake on the formation of new blood vessels. Our findings suggest that drugs designed to block RBPJ may promote new blood supplies and improve heart attack outcomes.”

 The authors also suggested that RBPJ itself might be beneficial in cancer if it can inhibit the new blood vessels tumors need to thrive.

 

Bionic patch as pacemaker.  Chemists at Harvard have designed nanoscale electronic scaffolds that can be seeded with heart cells and are able to conduct current to detect irregular heart rhythms and potentially send out electrical signals to correct them.

 “Rather than simply implanting an engineered patch built on a passive scaffold, our works suggests it will be possible to surgically implant an innervated patch that would now be able to monitor and subtly adjust its performance,” said Charles Lieber the senior author in a university press release posted by Phys.Org. The research was published in Nature Nanotechnology

 With its electronics built into the patch that is integrated into the heart, Lieber suggested the bionic patch could detect heart rhythm problems sooner than traditional pace makers. Another use for the patch he suggested could be to screen potential drugs.

 

Alternate to Tommy John in pictures. Sports fans generally have a vague idea of what Tommy John surgery is. First performed on baseball pitcher Tommy John of the LA Dodgers in 1974, the surgery replaces a torn elbow tendon with one from another part of the body.  A number of baseball players in the past couple years have made headlines because they sought out an alternative to this invasive procedure using stem cells.

The players sometimes improve, but with their high-priced team doctors also demanding extensive physical therapy and other interventions, we don’t really know how much of the improvement is due to the stem cells.  I am not aware of controlled clinical trials looking at the alternative therapy.

LA Angels Andrew HeaneyBut given how much it is in the news, I thought it would be good to share this excellent info-graphic from the LA Times explaining exactly what happens with the stem cell version of the Tommy John procedure. The Times posted the graphic yesterday, and then today, papers around the country ran stories that the most recent famous recipient of the cells, Los Angeles Angels lefthander Andrew Heaney, was going to have the old-fashioned surgery today because the stem cell treatment did not work in this case.

There may be some individuals, likely those with only partial tears who might benefit from this stem cell procedure that uses a type of stem cell that is not likely to replace tendons, but can release factors that summons the body’s natural healing apparatus to do a better job.  But until more formal clinical trials are conducted, it will be hard for     doctors to know who would and would not benefit.

Multi-Talented Stem Cells: The Many Ways to Use Them in the Clinic

CIRM kicked off the 2016 International Society for Stem Cell Research (ISSCR) Conference in San Francisco with a public stem cell event yesterday that brought scientists, patients, patient advocates and members of the general public together to discuss the many ways stem cells are being used in the clinic to develop treatments for patients with unmet medical needs.

Bruce Conklin, Gladstone Institutes & UCSF

Bruce Conklin, Gladstone Institutes & UCSF

Bruce Conklin, an Investigator at the Gladstone Institutes and UCSF Professor, moderated the panel of four scientists and three patient advocates. He immediately captured the audience’s attention by showing a stunning video of human heart cells, beating in synchrony in a petri dish. Conklin explained that scientists now have the skills and technology to generate human stem cell models of cardiomyopathy (heart disease) and many other diseases in a dish.

Conklin went on to highlight four main ways that stem cells are contributing to human therapy. First is using stem cells to model diseases whose causes are still largely unknown (like with Parkinson’s disease). Second, genome editing of stem cells is a new technology that has the potential to offer cures to patients with genetic disorders like sickle cell anemia. Third, stem cells are known to secrete healing factors, and transplanting them into humans could be beneficial. Lastly, stem cells can be engineered to attack cancer cells and overcome cancer’s normal way of evading the immune system.

Before introducing the other panelists, Conklin made the final point that stem cell models are powerful because scientists can use them to screen and develop new drugs for diseases that have no treatments or cures. His lab is already working on identifying new drugs for heart disease using human induced pluripotent stem cells derived from patients with cardiomyopathy.

Scientists and Patient Advocates Speak Out

Malin Parmar, Lund University

Malin Parmar, Lund University

The first scientist to speak was Malin Parmar, a Professor at Lund University. She discussed the history of stem cell development for clinical trials in Parkinson’s disease (PD). Her team is launching the first in-human trial for Parkinson’s using cells derived from human pluripotent stem cells in 2016. After Parmar’s talk, John Lipp, a PD patient advocate. He explained that while he might look normal standing in front of the crowd, his PD symptoms vary wildly throughout the day and make it hard for him to live a normal life. He believes in the work that scientists like Parmar are doing and confidently said, “In my lifetime, we will find a stem cell cure for Parkinson’s disease.”

Adrienne Shapiro, Patient Advocate

Adrienne Shapiro, Patient Advocate

The next scientist to speak was UCLA Professor Donald Kohn. He discussed his lab’s latest efforts to develop stem cell treatments for different blood disorder diseases. His team is using gene therapy to modify blood stem cells in bone marrow to treat and cure babies with SCID, also known as “bubble-boy disease”. Kohn also mentioned their work in sickle cell disease (SCD) and in chronic granulomatous disease, both of which are now in CIRM-funded clinical trials. He was followed by Adrienne Shapiro, a patient advocate and mother of a child with SCD. Adrienne gave a passionate and moving speech about her family history of SCD and her battle to help find a cure for her daughter. She said “nobody plans to be a patient advocate. It is a calling born of necessity and pain. I just wanted my daughter to outlive me.”

Henry Klassen (UC Irvine)

Henry Klassen, UC Irvine

Henry Klassen, a professor at UC Irvine, next spoke about blinding eye diseases, specifically retinitis pigmentosa (RP). This disease damages the photo receptors in the back of the eye and eventually causes blindness. There is no cure for RP, but Klassen and his team are testing the safety of transplanting human retinal progenitor cells in to the eyes of RP patients in a CIRM-funded Phase 1/2 clinical trial.

Kristen MacDonald, RP patient

Kristen MacDonald, RP patient

RP patient, Kristen MacDonald, was the trial’s first patient to be treated. She bravely spoke about her experience with losing her vision. She didn’t realize she was going blind until she had a series of accidents that left her with two broken arms. She had to reinvent herself both physically and emotionally, but now has hope that she might see again after participating in this clinical trial. She said that after the transplant she can now finally see light in her bad eye and her hope is that in her lifetime she can say, “One day, people used to go blind.”

Lastly, Catriona Jamieson, a professor and Alpha Stem Cell Clinic director at UCSD, discussed how she is trying to develop new treatments for blood cancers by eradicating cancer stem cells. Her team is conducting a Phase 1 CIRM-funded clinical trial that’s testing the safety of an antibody drug called Cirmtuzumab in patients with chronic lymphocytic leukemia (CLL).

Scientists and Patients need to work together

Don Kohn, Catriona Jamieson, Malin Parmar

Don Kohn, Catriona Jamieson, Malin Parmar

At the end of the night, the scientists and patient advocates took the stage to answer questions from the audience. A patient advocate in the audience asked, “How can we help scientists develop treatments for patients more quickly?”

The scientists responded that stem cell research needs more funding and that agencies like CIRM are making this possible. However, we need to keep the momentum going and to do that both the physicians, scientists and patient advocates need to work together to advocate for more support. The patient advocates in the panel couldn’t have agreed more and voiced their enthusiasm for working together with scientists and clinicians to make their hopes for cures a reality.

The CIRM public event was a huge success and brought in more than 150 people, many of whom stayed after the event to ask the panelists more questions. It was a great kick off for the ISSCR conference, which starts today. For coverage, you can follow the Stem Cellar Blog for updates on interesting stem cell stories that catch our eye.

CIRM Public Stem Cell Event

CIRM Public Stem Cell Event

Sickle Cell Disease Leaves No Organ Untouched

“There really isn’t an organ in the body that isn’t affected by sickle cell disease.”

This striking comment was made by the Dr. Bertram Lubin, the CEO and President of the Children’s Hospital Oakland Research Institute (CHORI) and a CIRM Board Member.

Yesterday Dr. Lubin visited CIRM headquarters to talk about sickle cell disease (SCD). SCD is a group of inherited disorders caused by unhealthy, sickle-shaped red blood cells. People with SCD have abnormal hemoglobin, an important protein in red blood cells used to transport oxygen from the lungs to organs and tissues throughout the body.

The What, Why and Who of SCD

A mutation in the globlin gene leads to sickled red blood cells that clog up blood vessels

A mutation in the globlin gene leads to sickled red blood cells that clog up blood vessels

Genetic mutations in the hemoglobin genes lead to changes in the hemoglobin protein that cause normal, healthy disc-shaped red blood cells to take on a crescent, sickle shape. These sickle cells are a big problem because they stick to each other and to the walls of blood vessels, causing blockage and impeding blood flow. This leads to a plethora of clinical complications that we will touch on later in this blog.

Dr. Lubin shared some shocking facts including that 2 million African Americans are carriers of SCD mutations and 100,000 Americans have the disease. In the US, 1000 babies are born with SCD each year, but this number pales in comparison to the 1000 African babies that are born with SCD each day.

“So anything we do here with CIRM has a direct impact on sickle cell disease,” Lubin explained. “It’s something we should consider because it could have a global impact on SCD.”

SCD Affects Every Organ in the Body

Dr. Bertram Lubin

Dr. Bertram Lubin

Dr. Lubin next discussed a laundry list of clinical manifestations associated with SCD, making it clear that SCD is not just a blood disorder, it affects every organ and tissue in the body. Examples he gave included infection, enlarged spleen, stroke, bone disease, retinopathy, and gastro-intestinal complications. And these were only a handful of the symptoms he discussed that SCD patients deal with.

However, Dr. Lubin emphasized that early detection of SCD in babies can drastically improve the quality and length of life of SCD patients. He proudly explained how California was the first state to screen every newborn baby for SCD (a procedure that is now done in every state) and that CHORI’s Center for Sickle Cell Disease and Thalassemia is one of the major SCD programs in the world. Their center “strives to improve public awareness of these diseases, expand the current knowledge base, and ultimately, to provide innovative treatment, care – and cures.”

Dr. Lubin also commented on the importance of knowing if patients who go to the ER or doctor have SCD:

Dr. Bertram Lubin

Dr. Bertram Lubin

“With new born screening before we identified who had sickle cell disease, an African American child could come to the emergency room with a 103 F temperature. And they would say, well this is a virus, go home, and half of those kids would die by the next day. Because those with pneumococcal sepsis [a bacterial infection that SCD patients have an increased risk for] don’t last very long. Now when someone comes into the emergency room with a 103 F temperature and we know they have sickle cell, they get antibiotics right away. That told us there is a different way to do it and that really showed how genetics and public health can have an impact on the overall health of the population.”

Treatments and Hope for SCD

Dr. Lubin ended his talk by discussing the current management and treatment strategies for SCD patients. Early identification through universal newborn screening and family education are essential as well as preventative measures like penicillin and immunization to avoid infection.

As for therapeutic interventions, he mentioned blood transfusions, hydroxyurea treatments (which boosts the levels of healthy hemoglobin in blood cells), and bone marrow stem cell transplants. He said while bone marrow transplants have successfully treated some SCD patients, there are still many barriers to this form of treatment. Only 14% of families of SCD patients have an HLA-identical sibling donor and only 19% have an unrelated HLA-matched donor. Additionally, some doctors avoid recommending bone marrow transplants to SCD patients because of the risks for transplant rejection (graft vs. host disease) and death.

However, Dr. Lubin is hopeful that recent advances in stem cell research and genome engineering will one day make stem cell transplants the go-to treatment for SCD patients.

He ended with:

“The future of curative therapies that will have broad availability for SCD might follow advances in genomic correction of sickle mutation in hematopoietic [bone marrow] stem cells.”


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Stem cell stories that caught our eye: reducing radiation damage, making good cartilage, watching muscle repair and bar coding cells

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.

A bomb blastaStem cells key to reducing radiation damage. With the anniversary of Hiroshima and President Obama’s historic visit to the site all over the news this week, it was nice to read about research that could result in many more people surviving a major radiation event—either from a power plant accident or the unthinkable repeat of history.

Much of the life-threatening damage that occurs early after radiation exposure happens in the gut, so a way to reduce that damage could buy time for other medical care. A team at the University of Texas Medical Branch at Galveston has discovered a drug that activates stem cells in the gut, which help maintain a healthy population of crypt cells that can repair gut damage.

A single injection of the small protein drug in mice significantly increased their survival, even if it was given 24 hours after exposure to radiation. The researchers published their work in the journal Laboratory Investigation and in a story written for MedicalNewsToday the lead author, Carla Kantara suggested the role the drug might have:

 “The current results suggest that the peptide may be an effective emergency nuclear countermeasure that could be delivered within 24 hours after exposure to increase survival and delay mortality, giving victims time to reach facilities for advanced medical treatment.”

The small protein, or peptide, named TP508, has already been tested in humans for diabetic foot ulcers so could be tested in humans fairly quickly.

 

Making good cartilage for your knees. Rarely a week goes by that I don’t tell a desperate osteoarthritis patient with painful knees that I am treating my own rotten knees with physical therapy until we learn how to use stem cells to make the right kind of cartilage needed for lasting knee repair. So, I was thrilled to read this week that the National Institutes of Health awarded Case Western Reserve University in Cleveland $6.7 million to develop a center to create standardized systems for monitoring stem cells as they convert into cartilage and for evaluating the resulting cartilage.

ear_wakeforest There are a couple problems with existing attempts to use stem cells for knee and other cartilage repair. First not all cartilage is equal and too often stem cells form the soft kind like in your earlobe, not the hard kind needed to protect knees. Also, it has been hard to generate enough cells to replace the entire area that tends to be eroded away in osteoarthritis, one of the leading causes of disability.

The new center, which will be available to researchers anywhere in the world, will develop tools for them to measure four things:

  • which genes are turned on or off as stem cells take the many steps toward becoming various forms of cartilage;
  • predict the best makeup of the extracellular matrix, the support structures outside cells that help them organize as they become a specific tissue;
  • evaluate the biochemical environment around the cells that helps direct their growth;
  • measure the mechanical properties of the resulting cartilage—is it more like the ear or the knee.

NewsWise posted the university’s press release

 

Damaged muscle grabs stem cells.  All our tissues have varying skills in self repair. Muscles generally get pretty high marks in that department, but we don’t really know how they do it. A team at Australia’s Monash University used the transparent Zebra fish and fancy microscopes to actually watch the process.

When they injured mature muscle cells they saw those cells send out projections that actually grabbed nearby muscle stem cells, which regenerated the damaged muscle. They published their findings in Science, the university issued a press release and a news site for Western Australia, WAtoday wrote a story quoting the lead researcher Peter Currie:

 “A significant finding is that the wound site itself plays a pivotal role in coordinating the repair of damaged tissue. If that response could be sped up, we are going to get better, or more timely, regeneration and healing.”

The online publication posted four beautiful florescent images of the cells in action.

 

muscle stem cells Monash

Muscle stem cells in action

“Bar coding” cells points to better transplants.  A team at the University of Southern California, partially funded by CIRM, developed a way genetically “bar code” stem cells so they can be tracked after transplant. In this case they watched the behavior of blood-forming stem cells and found the dose of cells transplanted had a significant impact on what the cells became as they matured.

The general dogma has blood stem cells producing all the various types of cells in our blood system including all the immune cells needed by cancer patients after certain therapies. But the USC tracking showed that only 20 to 30 percent of the stem cells displayed this do-it-all behavior. The type of immune cells created by the remaining 70 to 80 percent varied depending on whether there was a low dose of cells or a high dose, which can be critical to the effectiveness of the transplant.

 “The dose of transplanted bone marrow has strong and lasting effects on how HSCs specialize and coordinate their behavior,” said Rong Lu, senior author, in a USC press release posted by ScienceDaily. “This suggests that altering transplantation dose could be a tool for improving outcomes for patients — promoting bone marrow engraftment, reducing the risk of infection and ultimately saving lives.”

Stem cell stories that caught our eye: two-week old embryos in the lab, gene edited disease model, recipe for bone and cancer milestone

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.

Two-week embryos grabbed headlines. I have rarely seen as many online news outlets pick up a basic science story as happened this week with the news that an international team had nearly doubled the time it is possible to keep an embryo alive in a lab dish. While the research has tremendous potential to improve the chances couples can bring a new life into their families, the bulk of the coverage focused on the ethical issues surrounding the research embryo itself.

 

Imaris Snapshot

Molecular markers highlight various parts of a 12-day old embryo

After countless national and international confabs in the late 1970s and into the 1980s, research organizations around the world adopted the policy that no one would grow an embryo in the lab beyond 14 days. That is the point the “primitive streak” develops marking the first time cells within the embryo adopt individual identities. But the rule required no enforcement because no one knew how to coax an embryo into growing beyond nine days, and few could get them even to grow seven.

That changed this week, when the team led by Ali Brivanlou at Rockefeller University got embryos to grow to 13 days. They followed a procedure developed by a team colleague in Cambridge, UK, in mice reported earlier. They basically made the embryos feel more at home. They tested many different chemicals to add to the lab dish to optimize growth and gave the embryos a rigid structure more like a uterine wall.

They successfully mimicked implantation, the key step when the few-day-old embryo attaches to the uterus. Failure in this critical step is a key cause of infertility, but we have never been able to find out how it happens, and what little we do know suggests the mouse model for that step is not a good one for looking at human fertility.

 “This portion of human development was a complete black box,” said Brivanlou in a university press release picked up by many outlets including Bioscience Technology. She later added: “With this work, we can really appreciate the differences between human and mouse, and across all mammals. Because of the variations between species, what we learn in model systems is not necessarily relevant to our own development, and these results provide crucial information we couldn’t learn elsewhere.”

Because of that incredible potential value in this work, the journal Nature that published the research paper also ran a commentary about the current 14-day limit on growing embryos in the lab. It does not call for changing the policy at this time, but it does suggest the conversation–likely to be long–about whether the benefits of this work outweigh the ethical trip wires should begin soon.

The Washington Post wrote one of the most balanced pieces discussing both sides of the issue.

 

A mightier disease-in-a-dish model.  We frequently write about using iPS type stem cells to model diseases. Usually this involves getting a skin sample from a patient with a genetically-linked disease, converting it to stem cells and then growing the nerve or other tissue impacted by the disease. But you can also mimic the disease by genetically modifying normal stem cells to have specific mutations. This allows you to start to sorting out the role of individual genes in diseases linked to multiple genes.

 

Neurons from stem cells_TessierLavigne_neurons

Nerves grown from stem cells

One problem with the latter had been that gene editing techniques, particularly the wildly popular CRISPR-Cas9 method, usually edit both strands of DNA, but many disease mutations can do their damage with only a single incorrect gene, so-called heterozygous mutations. Now, another Rockefeller University team, this one led by the University’s president Marc Tessier-Lavigne, developed a way to make the CRISPR edit much more specific and only impact one strand of DNA.

HealthCanal picked up the university’s press release about the work published in Nature. The specific gene editing in this reports involved mutations linked to Alzheimer’s disease.

 

bone-scaffold Hopkins

Printed jaw

A better recipe for bone. Researchers trying to grow new tissue are finding the make-up of the scaffold you use can be more important than the stem cells you put on the structure. A Johns Hopkins team recently reported an improved recipe for making a scaffold for growing bone. Their formula: 30 percent pulverized natural bone and the remainder a special plastic with the mixture extruded using a 3D printer.

 “Bone powder contains structural proteins native to the body plus pro-bone growth factors that help immature stem cells mature into bone cells,” said Hopkins’ Warren Grayson. “It also adds roughness to the PCL (plastic), which helps the cells grip and reinforces the message of the growth factors.”

MDTmag posted the university press release about the research published in ACS Biomaterials Science & Engineering.

 

Licensing moved cancer therapy forward.  We at CIRM are always thrilled when one of our projects hurdles a milestone toward becoming a widely available therapy. One such critical move was announced last month and picked up this week by HealthCanal.

 Oncternal Therapeutics licensed the antibody drug named for our agency, Cirmtuzumab, for further testing of its ability to fight leukemia, and potentially other cancers. The antibody selectively targets a protein on cancer stem cells, ROR1, which has the unwieldy full name “receptor-tyrosine kinase-like orphan receptor 1.” The license also includes rights to other drugs that might be developed targeting ROR1.

University of California, San Diego, which developed Cirmtuzumab, has begun a clinical trial but has not got to a point where it can report results. We covered it in more detail in our series CIRM Fights Cancer.

In the Stem Cellar: restoring vision, a visionary on mini-organs and restoring circulation to limbs

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.

Eyes owned the web this week. When your work includes any form of electronic communication, you worry about how many eyes see a page of content. Well this week you could not be anywhere near the internet and not see lots of pages about the eye. Two studies published in Nature offered such great hope for restoring vision robbed by cataracts that no media outlet could resist covering these fairly complex and scientifically elegant papers.

In one paper, researchers announced they had corrected the vision of a dozen young children born with cataracts by enabling their own native stem cells in the eye. The team from the University of California, San Diego, the University of Texas Southwest Medical Center in Dallas, and Sun Yat-Sen University in Guangdong, China, said their procedure resulted in better vision than traditional transplantation of a donor lens for the clouded eye.

 “An ultimate goal of stem cell research is to turn on the regenerative potential of one’s own stem cells for tissue and organ repair and disease therapy,” said Kang Zhang of the UCSD team in a story in Times of San Diego.

 In the other research report a team from Osaka University in Japan and Cardiff University in the UK used iPS type stem cells to create complex tissues that mimicked several parts of the eye. One was the cornea, which they implanted into rabbits and restored their vision. Genetic Engineering News wrote the piece with the most detail on this study and in that article the team notes the procedure is not yet ready for humans but they are working on it.

The web site for CTV in Canada did one of the better pieces trying to talk about both studies. It included quotes from a commentary piece in the same issue of the journal by Julie Daniels of the University College London:

 “These two studies illustrate the remarkable regenerative and therapeutic potential of stem cells.”

 

organs-on-chips

Organs on chips at the Wyss Institute

The art of designing organs.  Last spring the Museum of Modern Art in New York added some “organs-on-chips” to its collection. That exhibit became the jumping off point for a commentary in a special issue of the journal Cell on the biology of communication. One of the leaders in applying systems approaches to bioengineering, Donald Ingber of the Wyss Institute at Harvard wrote the commentary, and a university press release got picked up by a few outlets including Medical News Today.

Ingber describes the organs on chips as a great way to analyze how organs function and how they fit in with the overall function of the body.  And he hopes they will eventually lead to replacement parts.

 “We’re not trying to rebuild a human organ,” said Ingber. “We’re trying to develop culture environments for living human cells with the minimal design features that will induce them to reconstitute organ level structures and functions to mimic the physiology that we see in the human body.”

He sees particular power in such mini organs when paired with stem cell technology.  In particular, he noted the potential to test drugs on a patient’s own mini-organ grown from their own genetically identical cells using iPS type stem cells.

 

Help for poor circulation in limbs.  Just by its name, critical limb ischemia sounds like something nasty that you do not want to have. Its root cause, blood vessel narrowing, usually in the legs, causes severe pain and often leads to amputation. Up to now efforts to use growth factors to create new blood vessels or to use donor stem cells to induce new vessels or replace muscle damaged by poor circulation have not worked.

Karen Christman

UCSD’s Karen Christman

Now, a team at the University of California, San Diego, is getting a “patient’s” own stem cells to do the job by giving them the right environment. The patients in this case are rats. They injected the damaged area with a gel made from the scaffold left behind when you wash the cells from muscle tissue. Many studies have shown that this extracellular matrix holds signals that can instruct stem cells and other cells to create appropriate tissues. It worked in the four-legged patients.

 “This is a unique approach that not only helps repair the damaged vascular system, but also helps restore muscle tissue,” said Karen Christman in a story posted by Scicasts adopted from a university press release.

To verify the improvement they analyzed which genes were activated in the area of the injections and showed that the inflammatory response was damped while blood vessel and muscle growth genes were activated.

Meet ITOP: A One Stop Shop for 3D Printing Body Parts

“They have managed to create what appears to be the goose that really does lay golden eggs!”

That was how UK surgeon Martin Birchall described it to BBC News. The goose in this case is a 3D bioprinter, and the golden eggs are the human sized tissues that the bioprinter successfully constructed. This breakthrough for the field of tissue engineering was reported on Monday in Nature Biotechnology by a research team led by Anthony Atala, director of Wake Forest Institute for Regenerative Medicine.

Bioprinting: yes, it’s actually a thing

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The 3D bioprinting process. Image: Wake Forest, Nature Biotechnology

To some, printing human body parts may sound like a far-fetched story torn from a science fiction novel but, in reality, development of bioprinters has been underway for a number of years. The bioprinting process isn’t all that different from an inkjet printer except a mixture of gelatin, or hydrogel, and living cells is used as the “ink” to incrementally build a defined  biological structure.

As amazing as this technology is, it has met some limitations. Printing cells into 2D shapes and small 3D structures is doable but a lack of structural stability limits building more complex, human-scale tissues. Also, because oxygen and nutrients can only diffuse about 0.004 inches through living tissue, the cells located inside a large bioprinted structure have trouble with long-term survival (check out yesterday’s blog for a mind-blowning story about one lab’s use of cotton candy to deal with this diffusion issue). These challenges have to be overcome before 3D bioprinting can be used for the repair and replacement of human-sized tissues and organs.

Enter ITOP
That’s where the Atala team’s Integrated Tissue-Organ Printer (ITOP), developed over ten years, comes into the picture. For better structural stability, the printer is configured to deliver the cell/hydrogel “bio-ink” within a stronger type of gel, with the fancy name Pluronic F127, that helps the printed cells maintain their shape during the printing process. Afterward, the Pluronic F127 scaffolding mold is simply washed away from the bioprinted tissue.

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Close-up view of ITOP: a 3D bioprinter. Image: Wake Forest, Nature Biotechnology.

To ensure adequate oxygen diffusion into the bioprinted tissue, a biodegradable polymer, PCL, is dispensed from the printer at regular intervals; this creates microchannels as stand-ins for blood vessels to help oxygen and nutrients readily reach the interior areas of the tissues.  As a bonus, PCL takes about 2 years to biodegrade, providing long term stability.

To prove these innovations of the ITOP actually work, the team built three different tissues: a jawbone fragment, an ear, and muscle.

The making of a jawbone
For reconstruction of the jawbone, a 3D computer model was generated from actual CT scan data of a human jaw with a missing piece of bone – something that might be seen in a traumatic injury to a combat soldier (the work is funded in part by the Armed Forces Institute for Regenerative Medicine). That data was fed into the ITOP with coordinates of the precise printing pattern necessary to rebuild the shape of the jaw fragment. In this case, printing was carried out using human amniotic fluid stem cells (AFSC). With the right cues, these stem cells readily specialize into osteogenic, or bone-forming, cells.

Sure enough, 28 days after being cultured in liquid nutrients containing bone-promoting factors, the surface of the bioprinted human jaw showed calcium deposits, the tell-tale signs of bone formation. How would bioprinted bone fare in a living mammal? To find out, the researchers transplanted small discs of AFSC fabricated bone into a bone defect in mice. After 5 months, the transplanted bone was thriving with plenty of blood vessels and no necrosis, or cell death, inside the bone.

The implications of this bone study are pretty cool. In an interview with BBC news, Atala envisions a not so distant future clinical scenario:

“We’d bring the patient in, do the imaging and then we would take the imaging data and transfer it through our software to drive the printer to create a piece of jawbone that would fit precisely in the patient.”

Vincent van Gogh could have used this technology
But the possibilities don’t end with bone. Next, the team tested the ITOP’s talents at building the complex shapes of the outer human ear. In this case, the printer was loaded up with cartilage-producing cells called chondrocytes. The authors posted a fascinating video of the ear bioprinting process in their online publication.

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A human-sized bioprinted ear. Image: Wake Forest, Nature Biotechnology

Image: Wake Forest, Nature Biotechnology.

After five weeks in liquid nutrients, a matrix of cartilage had grown throughout the ear. And to look at tissue growth in an animal, the ear was implanted under the skin of the mice. A couple of months after implantation, even more cartilage had formed and the shape of the ear was intact.

But wait there’s more: printing skeletal muscle
Since both the jaw bone and ear cartilage represent hard tissues, the team sought to reconstruct muscle, a soft tissue, with the ITOP. Muscle-forming cells, or myoblasts, were printed to mimic the muscle fiber bundles seen in native skeletal muscle. After growing a week in the lab under conditions that stimulate muscle cell formation, the muscle-like fibers were implanted into rats. Two week after implantation, the bioprinted muscle had not only grown into well-organized muscle fibers, they also were functional in that they were responsive to electrical stimulation.

3D bioprinting: getting closer to reality
Atala is the first to admit that a lot more testing is needed to safely bring this technology into a clinical setting for human use. But as he states in a Wake Forest press release, the ITOP brings 3D bioprinting a step closer to reality:

“This novel tissue and organ printer is an important advance in our quest to make replacement tissue for patients. It can fabricate stable, human-scale tissue of any shape. With further development, this technology could potentially be used to print living tissue and organ structures for surgical implantation.”

 

 

Stem cell stories that caught our eye: heart muscle-on-a-chip, your own private microliver, the bloody holy grail and selfish sperm

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.

Two hearts beat as one, or not
Sorry for the pre-Valentine’s Day buzzkill but stem cell research published this past week points to a very unromantic discovery: two hearts do not beat as one. The study, out of Rockefeller University, and published in the Journal of Cell Biology, sought to understand the limited success of clinical trials in which stem cell-derived heart muscle cells, or cardiomyocytes, are transplanted into the heart to help repair tissue scarred by disease or a heart attack.

If you’re a regular at The Stem Cellar, you’ll recall that just last Friday we summarized published experiments that suggest the cardiomyocytes used in successful trials do not grow new tissue themselves but instead heal the heart indirectly by releasing proteins that stimulate repair.

The research team behind this week’s study instead reasoned that the transplanted cardiomyocytes do indeed integrate into the heart tissue, but they fail to contract properly with the undamaged heart cells. So, the thinking goes, the transplanted cells do nothing to restore the heart’s ability to beat at full strength.

Watch video here: http://medicalxpress.com/news/2016-02-muscles-on-a-chip-insight-cardiac-stem.html

A two-cell “microtissue” contains a mouse embryonic stem cell-derived cardiomyocyte and a mouse neonatal cardiomyocyte. The lower panel shows the traction forces generated as the two cells contract; the stronger, neonatal cardiomyocyte produces more force than the weaker, stem cell-derived cardiomyocyte. Credit: Aratyn-Schause, Y. et al. J Cell Biol. 2016 Watch video here: http://medicalxpress.com/news/2016-02-muscles-on-a-chip-insight-cardiac-stem.html

 

To test this hypothesis, the researchers devised a two-cell micro-tissue made up of a single mouse cardiomyocyte and a single cardiomyocyte derived from either mouse embryonic stem cells or induced pluripotent stem cells (iPS). This “muscle-on-a-chip” showed that the two cells are able to physically connect up and even beat in sync with each other. But, the embryonic and iPS-derived cardiomyocytes beat less strongly than the native cell. Based on computer simulations, this imbalance made the micro-tissue beat less efficiently. A university press release picked up by Newswise includes a short yet fascinating video of the differing strengths of the beating heart cells (click on image above).

With this micro-tissue in hand, the team aims to find a way to fix this imbalance, which hopefully would make cell therapies for heart disease more potent.

Your Own Private Micro-liver
Enough about micro-hearts, let’s talk micro-livers.

In a report published on Monday in PNAS, a multidisciplinary UCSD team of engineers and biomedical researchers described the creation of a bioprinted 3D liver model made from human iPS-derived liver cells, or hepatocytes. The hepatocytes are imprinted on a surface in hexagonal shapes, the kind seen in the complex microarchitecture of the human liver. These structures were also seeded with two other cell types: endothelial cells, which form blood vessels, and fat cells, which support the health of hepatocytes. Including these relevant cell types in the “micro-liver” design resulted in a 3D cell culture that not only mimics structures but also replicates functions found in a natural liver.

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The 3-D-printed parts of the biomimetic liver tissue include: liver cells derived from human induced pluripotent stem cells (left), endothelial and mesenchymal supporing cells (center), and the resulting organized combination of multiple cell types (right). — Chen Laboratory, UC San Diego

This is a really exciting development for improving drug safety. A big concern of any new drug coming on the market is its potential liver toxicity, formally known as DILI (drug induced liver injury), the most common cause of liver failure in the U.S. Although animal studies and clinical trials carefully test for the potential of DILI, that doesn’t guarantee the drug will be safe in all individuals. And because this liver model was designed using human iPS cells – which can be derived from anyone with a simple skin biopsy – it has the potential to serve as a personalized drug screening device as well as a disease-in-a-dish model for studying inherited forms of liver disease.

As Bradley Fikes, San Diego Union Tribune’s biotechnology writer, mentions in an excellent summary of the publication, beyond drug screening and disease-in-dish modeling, this bioprinting process could also one day make it possible for researchers to reach the “holy grail” of tissue engineering: building an entire organ.

Finally! The Bloody Holy Grail
While that holy grail remains on the horizon, Stanford researchers are nearly holding the goblet in their hands. Based on a Nature report published yesterday, a team led by CIRM grantee Irv Weissman have found a long sought after cellular tag that can fish out a very specific type of hematopoietic stem cell (HSC), or blood-forming stem cell, from bone marrow.

Almost thirty years ago, Weissman identified HSCs, which have the ability to form all the cell types of the blood. Since that time, scientists have struggled with fully understanding how HSCs are maintained in the body and, in turn, how to grow them in the laboratory.

The source of this problem is due to the fact that most HSCs are so-called short term HSCs because they eventually lose their “stemness”; that is, their ability to divide indefinitely. Only a small fraction of HSCs are of the long-term variety. To really understand how the body sustains a life-long supply of HSCs, it’s necessary to have a method to pick out just the long term HSCs.

So scientists in Weissman’s lab set out to do just that. Starting with a list of 100 genes that are known to be active in the bone marrow, they looked for genes that are turned on only in long term HSCs. After a painstaking, systematic method that took two years, the team narrowed down the list to just one gene that was unique to long term HSCs.

Co-lead author James Y. Chen, a MD/PHD candidate at Stanford, described the significance of this effort in a university press release:

chen

James Y. Chen

“For nearly 30 years, people have been trying to grow HSCs outside the body and have not been able to do it — it’s arguably the ‘holy grail’ in this field. Now that we have an anchor, a way to look at long-term HSCs, we can look at the cells around them to understand and, ideally, recreate the niche.”

 

 

 

Older Dads and The Selfish Sperm

We wrap up the week with a PNAS publication that got a wide range of coverage by the likes of BBC News, Gizmodo and Cosmos in addition to the usual suspects like Health Canal. Not too surprising given the topic including selfish sperm and chopped up testicles.

Research over the past decade or so has made it increasingly clear that biological clocks not only tick for would-be moms but also dads. At first glance, it makes sense: older fathers have had more time to accumulate random DNA mutations in their spermatogonia, the stem cells that produce sperm. But studies of Apert syndrome, a rare disease causing defects in the skull, fingers and toes, has put this hypothesis in question.

Back in 2003, a research team at Oxford University found the mutation in spermatogonia that causes Apert syndrome occurs 100 to 1000 times more frequently than would be expected if it were merely due to a random mutation (the Apert syndrome is not inherited because males with the disease rarely go on to have children).

So what’s going on? To answer that question the Oxford scientists collaborated with a USC research team who (men: you may not want to read the rest of this sentence, this is your only warning) chopped up human testicles – ones that had been removed for unrelated medical reasons and donated – in order to reconstruct a three-dimensional map of where these Apert syndrome mutations were occurring. If the mutations were merely random, the affected spermatogonia would have been evenly distributed throughout the testicle. Instead, the team found clusters of cells carrying the mutation.

This results confirms a “selfish sperm” hypothesis in which the mutation provides a selective advantage to the affected sperm cells allowing them to out compete other nearby sperm cells, much like a cancer cell that multiples and gradually forms a tumor. The study serves as more sobering news to otherwise healthy older dads that they may have a higher risk of passing on harmful mutations to their offspring.

Like I said, sorry for the buzzkill. Happy Valentine’s Day weekend!