Fat-Based Mesenchymal Stem Cell-Seeded Matrix Heals Bronchopleural Fistula in Female Cancer Patient

Bronchopleural fistulae, mercifully abbreviated as BPF, refers to an opening or hole in the respiratory tree that causes continuity between the pleural space that surrounds the lungs and the bronchial tree. BPH is a highly feared complication of surgery on the respiratory system.

BPH can complicate surgical resection of the pulmonary system. Patients with lung cancers may require lung resection in order to remove tumorous lung tissue. The rate of BPH incidence after lung surgery varies widely, with reported incidences ranging from 1.5 to 28%. Necrosis or death of lung tissue as a result of infection can also cause BPH, as can tuberculosis. Chemotherapy or radiation therapy for lung cancers can also result in BPF. Finally, BPF may caused by persistent spontaneous pneumothorax, which refers to an abnormal build up of air or other gases in the pleural space, which causes an uncoupling of the lung from the chest wall.

To date, treatment for BPF is only partially effectively. The main treatment includes surgery, but the rate of recurrence of the fistulae remains rather high as do the rate of mortality. Can stem cells show us a better way?

Perhaps they can. Dennis A. Wigle, a surgeon at Mayo Clinic, and his collaborators used a synthetic bioabsorbable matrix seeded with the patients one fat-based mesenchymal stem cells to heal a BPF in a 63-yr old woman. Mind you, this is a case study (the lowest quality clinical evidence) and not a controlled study,. However, the success of this case study is at least suggestive that such an approach might prove useful for patients who suffer from BPFs.

Microscopic assessment of matrix cell seeding. (A): Ethidium bromide (red) and Syto-13 (green) costain demonstrating live and dead cells on mesenchymal stem cell seeding on matrix. (B): Confocal microscopy with CD90 (Thy-1) fluorescein isothiocyanate (green) and Hoechst 33342 (trihydrochloride trihydrate) (blue) fluorescent nuclear staining. These images were captured using a ×20 objective and a ×10 eyepiece, for a combined magnification of ×200. Scale bar = 150 µm.
Microscopic assessment of matrix cell seeding. (A): Ethidium bromide (red) and Syto-13 (green) costain demonstrating live and dead cells on mesenchymal stem cell seeding on matrix. (B): Confocal microscopy with CD90 (Thy-1) fluorescein isothiocyanate (green) and Hoechst 33342 (trihydrochloride trihydrate) (blue) fluorescent nuclear staining. These images were captured using a ×20 objective and a ×10 eyepiece, for a combined magnification of ×200. Scale bar = 150 µm.

A 63-yr old woman who had surgical resection of the lung in order to treat her lung cancer had, as a consequence of her surgery, a BPF. Some 30 different surgical attempts were made to repair the BPF, but all of them failed. The woman’s health declined and her medical team started to think of alternative treatments.

Fortunately, Mayo Clinic has been participating in an ongoing clinical trial to use fat-based mesenchymal stem cells to treat anal fistulae in Crohn’s disease patients. Therefore Dr. Wigle and his team considered using the protocol utilized with Crohn’s patients to repair this woman’s BPF.

Fat biopsies were taken from the patient and the fat was washed, minced, digested with enzymes, and then grown in special culture media. The adipose tissue-derived mesenchymal stem cells (AD-MSCs) grew and were isolated, characterized and shown to be MSCs.

These cells were then seeded on a matrix of synthetic bioabsorbable poly(glycolide-trimethylene carbonate) copolymer and then placed in a bioreactor to grow. After about 4 days, the matrix was flush with AD-MSCs, and this cell-seeded patch was then used in a subsequent surgery to seal the opening in the respiratory tree. This time the surgery worked. The patient was discharged 25 days after the surgery and sent home.

MRIs of the respiratory system showed that the BPF had indeed closed and properly resolved.

Preoperative imaging showing size and location of fistula, and postoperative imaging demonstrating disease resolution. (A): Preoperative bronchoscopy demonstrating large bronchopleural fistula (BPF) cavity and lateral extension of fistula tracts. (B): Postoperative bronchoscopy (3 months) demonstrating progressive healing of BPF site. (C): Preoperative computed tomography scan demonstrating large BPF with connection to atmosphere (additional axial slices inferiorly). (D): Postoperative computed tomography scan (16 months) demonstrating resolution of BPF.
Preoperative imaging showing size and location of fistula, and postoperative imaging demonstrating disease resolution. (A): Preoperative bronchoscopy demonstrating large bronchopleural fistula (BPF) cavity and lateral extension of fistula tracts. (B): Postoperative bronchoscopy (3 months) demonstrating progressive healing of BPF site. (C): Preoperative computed tomography scan demonstrating large BPF with connection to atmosphere (additional axial slices inferiorly). (D): Postoperative computed tomography scan (16 months) demonstrating resolution of BPF.

This case study might confirm what was previously observed in large animal studies by Petrella and others, namely that AD-MSCs can be used to heal BPF. Petrella and others theorized that implanted MSCs induce the proliferation of fibroblasts that then deposit collagen, which seals the BPF (see Ann Thorac Surg 97:480483.  Alternatively, AD-MSCs might differentiate into cell types  required for regeneration of the airways (Dominici M, and others, Cytotherapy 8:315317).  Either way, this paper seems to suggest that AD-MSCs can be isolated from a patient’s fat (even a very sick patient like this one) without incident and used for tissue engineering applications that can repair very serious wound like BPF. 

This paper was published in: Johnathon M., Aho, et Al., “Closure of a Recurrent Bronchopleural Fistula Using a Matrix Seeded With Patient-Derived Mesenchymal Stem Cells.” Stem Cells Trans Med October 2016 vol. 5 no. 10 1375-1379. 

Scientists Grow New Diaphragm Tissue In Laboratory Animals

The diaphragm is a parachute-shaped muscle that separates the thoracic cavity from the abdominopelvic cavity and facilitates breathing. Contraction of the diaphragm increases the volume of the lungs, thus dropping the pressure in the lungs below the pressure of the surrounding air and causing air to rush into the lungs (inhalation). Relaxation of the diaphragm decreases the volume of the lungs and increases the pressure in the lungs so that it exceeds the pressure of the air, and air leaves the lungs (exhalation). The diaphragm is also important for swallowing. One in 2,500 babies are born with malformations or perforations in their diaphragms, and this condition is usually fatal.

The usual treatment for this condition involves the construction of an artificial patch that properly covers the lesion, but has no ability to grow with the infant and is not composed of contractile tissue. Therefore, it does not assist in contraction of the diaphragm to assist in breathing.

A new study might change the prospects for these newborn babies. Tissue engineering teams from laboratories in Sweden, Russia and the United States have successfully grown new diaphragm tissue in rats by applying a mixture of stem cells embedded in a 3D scaffold made from donated diaphragm tissue. Transplantation of this stem cell/diaphragm matrix concoction into rats allowed the animals to regrow new diaphragm tissue that possessed the same biological characteristics as diaphragm muscle.
The techniques designed by this study might provide the means for repairing defective diaphragms or even hearts.

Doris Taylor, who serves as the director of regenerative medicine research at the Texas Heart Institute and participated in this revolutionary study, said: “So far, attempts to grow and transplant such new tissues have been conducted in the relatively simple organs of the bladder, windpipe and esophagus. The diaphragm, with its need for constant muscle contraction and relaxation puts complex demands on any 3D scaffold. Until now, no one knew whether it would be possible to engineer.”

Paolo Macchiarini, the director of the Advanced Center for Regenerative Medicine and senior scientist at Karolinska Institutet, who also participated in this study, said: “This bioengineered muscle tissue is a truly exciting step in our journey towards regenerating whole and complex organs. You can see the muscle contracting and doing its job as well as any naturally grown tissue.”

To make their tissue engineered diaphragms, the team used diaphragm tissue that had been taken from donor rats. They stripped these diaphragms of all their cells, but maintained all the connective tissue. This removed anything in these diaphragms that might cause the immune systems of recipient animals to reject the implanted tissue, while at the same time keeping all the things that give the diaphragm its shape and form. In the laboratory, the decellularized diaphragms had lost all their elasticity. However, once these diaphragm matrices were seeded with bone marrow-derived stem cells and transplanted into recipient laboratory animals, the diaphragm scaffolds began to function as well as normal, undamaged diaphragms.

If this new technique can be successfully adapted to human patients, it could replace the damaged diaphragm tissue of the patient with tissue that would constantly contract and grow with the child. Additionally, the diaphragm could be repaired by utilizing a child’s own stem cells. As a bonus, this technique might also provide a new way to

Next, the team must test this technique on larger animals before it can be tested in a human clinical trial.

The study was published in the journal Biomaterials.

3D Printing of Stem Cells on Bioceramic Molds to Reconstruct Skulls

Skull defects or injuries can be very difficult to repair. However, an Australian research team has pioneered a new technique that can regrow skulls by applying stem cells to a premade scaffold with a 3D printer.

This research team consists of a surgeon, a neurosurgeon, two engineers, and a chief scientist. This five-person team is collaborating with a 3D printing firm that is based in Vienna in order to manufacture exact replicas of bone taken from the skulls of patients.

The protocol for this procedure utilizes stem cells and 3D printers, and is funded by a $1.5 million research grant that is aimed at reducing costs and improving efficiency of the Australian public health service.

The first subjects for this procedure will include patients whose skulls were severely damaged, or had a piece of their skull removed for brain surgery, and require cranial reconstruction. The skull reconstructions will take place at the Royal Perth Hospital. The first trial will commence next year. If this procedure proves to be successful it could reduce the risk of complications and surgical time, and provide massive cost savings.

If a patient has a skull injury or some other skull issue, pieces of skull bone were removed bone and stored it in a freezer for later implantation into the skull. Unfortunately, this procedure often resulted in infection or resorption of the bone. Alternatively, titanium plates can be used but these eventually they degrade, and therefore, are not ideal.

Neurosurgeon Marc Coughlan, who is a member of the five-person research team that developed this procedure, said this protocol represents the first time stem cells have been used on a 3D printed scaffold to regrow bone. “What we’re trying to do is take it one step further and have the ceramic resorb and then be only left with the patient’s bone, which would be exactly the same as having the skull back,” Coughlan told The Australian.

If this procedure proves successful, it could revolutionize cranial reconstruction surgeries. According to health minister Kim Hames, “This project highlights some of the innovative and groundbreaking research that is under way in WA’s public health system, and the commitment of the government to supporting this crucial work.”

We will keep tabs on this clinical trial to determine if it works as well as reported.

Laboratory-Grown Intestine Shows Promise in Mice and Dogs

David Hackam is a pediatric surgeon at the Johns Hopkins Children’s Center. Unfortunately, Dr. Hackam spends a good deal of his time removing dead sections of intestine from sick babies, but he would deeply love to be able to do more than just take out intestines but actually replace the dead or dying intestinal tissue. It is that desire that has driven Hackam and his colleagues to grow intestines in the laboratory.

They begin with stem cells taken from the small intestines of human infants and mice and apply them to intestine-shaped scaffolds. The stem cells dig in, grow and form mini-intestines that just might be able to treat disorders like necrotizing enterocolitis and Crohn’s disease someday. Transplantation experiments in laboratory animals have shown that this laboratory-grown tissue and scaffolding are not rejected, but integrate into the tissues of the animals. Experiments in dogs have shown that the scaffold allowed dogs to heal from damage to the colon lining, essentially restoring healthy bowel function.

The study is a “great breakthrough,” says Hans Clevers, a stem cell biologist at the Hubrecht Institute in Utrecht, the Netherlands, who was not involved in the new research. Clevers and his colleagues were the first to identify stem cells in the intestine, and his lab developed the technique Hackam’s team used to grow intestinal tissue.

Making replacement organs by growing cells on scaffolds molded into the shape of the organ is not a new idea, since other researchers have used exactly this technique to make bladders and blood vessels. However, the laboratory-grown intestines made by Hackam and his group come closer to the shape and structure of a natural intestine than anything created in the laboratory before. In previous experiments carried out in other laboratories, the gut lining has been grown on flat scaffolds or in culture flasks. Under these conditions, the tissue tends to roll up into little balls that have the absorptive surface on the inside. Hackam and his coworkers, however, overcame this problem by using a scaffold fabricated from materials similar to surgical sutures. This material can be molded into any desired intestinal size and shape, and in Hackam’s hands, the scaffolds formed a true tube-shaped (like a real gut), with tiny projections on the inner surface that can help the tissue form functional small intestinal villi (the small fingers of tissue that increase the surface area of the intestine to increase nutrient absorption. “They can now make sheets of cells that can be clinically managed,” Clevers says. “Surgeons can handle these things and just stick them in.”

To grow the gut lining in the lab, the researchers painted the scaffold with a sticky collagen-rich substance and then dripped onto it a solution of stem cells from the small intestine. This concoction was grown in a culture system for a week. Interestingly, Hackam and his team found that if they added connective tissue cells, immune cells, and probiotics (bacteria that help maintain a healthy gut), all of these things helped the stem cells mature and differentiate.

Hackam’s group also sutured intestines grown from mouse stem cells into the tissue surrounding the abdominal organs of the mouse. The lab-grown intestines developed their own blood supply and normal gut structures despite the fact that they were not connected to the animals’ digestive tract. “Using the mouse’s own stem cells, we can actually create something that looks just like the native intestine,” Hackam says. The next step, he says, is “to hook it up.”

Before “hooking it up,” Hackam needed to be sure that the scaffold could be tolerated in living animals. Therefore he tested the new scaffold in dogs. He removed sections of large intestinal lining and replaced it with pieces of scaffolding. The dogs made a complete recovery: their gut lining regrew onto the scaffold and functioned normally to absorb water from the colon. After a few weeks, the scaffolding had completely dissolved and was replaced with normal connective tissue. “The scaffold was well tolerated and promoted healing by recruiting stem cells,” Hackam says. “[The dogs] had a perfectly normal lining after 8 weeks.”

This technique could help more than just dogs and mice, but could aid human patients. According to Hackam, scaffolds could be custom-designed for individual human patients to replace a portion of an intestine or the entire organ. This could be a revolutionary treatment for patients with necrotizing enterocolitis, a condition that destroys intestinal tissue in about 12% of premature babies in the United States. It could also potentially repair the intestines of patients with Crohn’s disease, an inflammatory bowel disorder that can have life-threatening complications and that affects more than 500,000 people in the United States. However, these lab-grown intestines must pass several other tests before they are ready for human clinical trials, Hackam cautions.

The first test that these laboratory-grown intestines must pass is the absorption test. Laboratory-grown small intestines must be transplanted into live animals and they must properly absorb food. Also, the technology that is used will also require some adjustments. For example, Mari Sogayar, a molecular biologist at the University of São Paulo in Brazil, points out that the collagen product that helps the stem cells stick to the scaffold is not meant for use in people. In the next experiments, Hackam says, the researchers plan to use a surgical-grade alternative.

“I take care of children who have intestinal deficiencies, eating deficiencies, and they are very much at wits’ end,” Hackam says. “I think what we can offer in the scientific community is a path toward something that one day will help a child.”

Hydrogels Help Implanted Stem Cells Survive in the Heart

How do you get stem cells to survive after they have been transplanted? You can pre-condition them, but research from Johns Hopkins University has capitalized on a different strategy. The Hopkins team used hydrogel to protect and feed the stem cells that had been implanted into the heart.

They utilized a rat model system for this work. Rats that had been given heart attacks were given stem cell implants encased in a hydrogel. The hydrogel supported stem cells survival and also kept the stem cells at the site of their implantation where they re-muscularized the damaged heart muscle. 73% of the stem cells embedded in hydrogel survived whereas only 12% of the non-hydrogel-embedded stem cells survived after injection into the heart.

Previously, stem cell injections have been shown to aid damaged heart tissue, but the vast majority of the injected cells die or are washed from the heart into other tissues. Hydrogel, which mostly consists of water, allows the cells to live and grow while they integrate into the surrounding tissue and initiate healing.

Heart-damaged rats injected with hydrogel-loaded stem cells saw a 15% increase in pumping efficiency for the treated ventricle, compared with just 8% for regular stem cell therapies. Hydrogen can support both adult and embryonic stem cells, and if it’s not put inside a living being, the hydrogel can actually maintain 100% of the stem cells embedded in them.

Hydrogels are useful in biology because they are safe for use in living organisms. In fact, this study found that injecting the hydrogel alone, with no stem cells at all, had a mild benefit all its own by promoting new blood vessel growth.

These are the sorts of breakthroughs that will allow the stem cell technologies of today to become the amazing stem cell technologies of tomorrow.

The Ideal Recipe for Cartilage from Stem Cells

Researchers at Case Western Reserve and Harvard University will use a 5-year, $2-million NIH grant to build a microfactory that bangs out the optimal formula for joint cartilage. Such an end product could one day potentially benefit many of the tens of thousands of people in the United States who suffer from cartilage loss or damage.

Joint cartilage or articular cartilage caps the ends of long bones and bears the loads, absorbs shocks and, in combination with lubricating synovial fluid, helps knees, hips, shoulders, and other joints to smoothly bend, lift, and rotate. Unfortunately, this tissue has little capacity to regenerate, which means that there is a critical need for new therapeutic strategies.

Artificial substitutes cannot match real cartilage and attempts to engineer articular cartilage have been stymied by the complexities of directing stem cells to differentiate into chondrocytes and form the right kind of cartilage.

Stem cells are quite responsive to the environmental cues presented to them from their surroundings. What this research project hopes to determine are those specific cue that drive stem cells to differentiate into chondrocytes that make the right kind of cartilage with the right kind of microarchitecture that resembles natural, articular cartilage. To do this, they will engage in a systematic study of the effects of cellular micro-environmental factors that influence stem cell differentiation and cartilage formation.

Bone marrow- and fat-derived mesenchymal stem cells have been differentiated into cartilage-making chondrocytes in the laboratory. These two stem cell populations are distinct, however, and required different conditions in order to drive them to differentiate into chondrocytes. This research group, however, has designed new materials with unique physical properties, cell adhesive capabilities, and have the capacity to deliver bioactive molecules.

By controlling the presentation of these signals to cells, independently and in combination with mechanical cues, this group hopes to identify those most important cues for driving cells to differentiate into chondrocytes.

Ali Khademhosseini specializes in microfabrication and micro-and nano-scale technologies to control cell behavior. He and his team will develop a microscale high-throughput system at his laboratory that will accelerate the testing and analysis of materials engineered in another laboratory.

This research cooperative hopes to test and analyze more than 3,000 combinations of factors that may influence cell development, including differentiation, amounts of biochemicals, extracellular matrix properties, compressive stresses, and more. Khademhosseini and his colleagues hope to begin testing comditions identified from these studies in animal models by the of the grant term.

Porous Hydrogels Boost Stem Cell-Based Bone Production

Regenerative medicine relies upon the ability to isolate, manipulate, and exploit stem cells from our own bodies or from the bodies of stem cell donors. A present obstacle to present therapeutic strategies is the poor survival of implanted stem cells. There are also worries of about properly directing the differentiation of transplanted stem cells. After all, if implanted stem cells do not differentiate into the terminal cell types you want to be replaced, the use of such cells seems pointless.

To address this problem, David Mooney from the Wyss Institute and his colleagues have designed a three-dimensional system that might keep transplanted stem cells alive and happy, ready to heal.

Mooney’s group has adopted a strategy based on the concept of stem cell “niches.”. In our bodies, stem cells have particular places where they live. These stem cell-specific microenvironments provide unique support systems for stem cells and typically include extracellular matrix molecules to which stem cells attach.

Mooney and others have identified chemical and physical cues that act in concert to promote stem cell growth and survival. The chemical cues found in stem cell niches are relatively well-known but the physical and mechanical properties are less well understood at the present time.

Stem cells in places like bone, cartilage, or muscle, when cultured in the laboratory, display particular mechanical sensitivities and they must rest on a substrate with a defined elasticity and stiffness in order to proliferate and mature. As you might guess, reproducing the right physical properties in the laboratory is no mean feat. However, several laboratories have used hydrogels to generate the right combination of chemical and physical properties.

Mooney and his colleagues have made two hydrogels with very different properties. A stable, “bulk” gel is filled with small bubbles of a pore-making molecule called a “porogen,” which degrades quickly and leaves porous cavities in its wake. When the bulk hydrogel is combined with extracellular matrix molecules from stem cell niches and filled with tissue-specific stem cells, and the porogen, Mooney and his team can make an artificial bone-forming stem cell niche. The porous cavities in the hydrogel, in combination with the chemical signals, drive the stem cells to grow, and divide while expanding into the open spaces in the gel. Then the cell move from the hydrogel to form mineralized bone.

In small animal experiments, Mooney and his colleagues showed that a porous hydrogel with the correct chemical and elastic properties provides better bone regeneration than transplanting stem cells alone. The maturing stem cells deployed by the hydrogel also induce neighboring stem cell populations to contribute to the bone repair, which further amplifies their regenerative effects.

This study provides the first demonstration that adjusting the physical properties of a biomaterial can not only help deliver stem cells but also tune the behavior of those cells in a living organism. Even though Mooney has primarily focused on bone formation, he and his group believe that the hydrogel concept can be broadly applied to other regenerative process as well.

This work was published in Nature Materials 2015; DOI: 10.1038/nmat4407.

Stem-Cell Dental Implants Grow New Teeth in Your Mouth

Dr. Jeremy Mao is the Edward V. Zegarelli Professor of Dental Medicine at Columbia University Medical Center. Mao and his colleagues have published a novel technology that includes a growth factor-infused, three-dimensional scaffold that has the potential to regenerate an anatomically correct tooth in the mouth just nine weeks after implantation. By this procedure, which was developed in the university’s Tissue Engineering and Regenerative Medicine Laboratory, Mao can direct the body’s own stem cells to migrate to the scaffold and infiltrate it. Once these stem cells have colonized the scaffold, they will produce a tooth that can grow in the socket and merge with the surrounding tissue and integrate into it.

Tooth scaffold that is completely composed of natural materials.
Tooth scaffold that is completely composed of natural materials.

Mao’s technique not only eliminates the need to grow teeth in a culture, but it can regenerate anatomically correct teeth by using the body’s own resources. If you factor in the faster recovery time and the comparatively natural process of regrowth (as opposed to implantation), you have a massively appealing dental treatment.

Columbia University has already filed patent applications in regard to this technology. They are also seeking associates to aid in its commercialization. Mao is also considering the best approach for applying his technique to cost-effective clinical therapies.

Laboratory-Grown Kidneys Work in Laboratory Animals

A Jikei University School of Medicine research team based in Tokyo, Japan, led by Takashi Yokoo, in collaboration with scientists from Meiji University and St. Marianna University School of Medicine in Kawasaki, Keio University School of Medicine in Tokyo, and the School of Veterinary Medicine at Kitasato University in Towada, has shown that mini kidneys grown in vitro from human stem cells can be effectively connected to the excretory systems of rats and pigs.

This is not the first time that research groups have successfully grown mini kidneys in the laboratory. However, connecting these laboratory-grown organs to a laboratory animal’s excretory system constitutes a major technical challenge. The Jikei University team used an approach that employs a step-wise peristaltic ureter or SWPU to connect its lab-grown mini kidneys to the ureter of the transplant animal.

Previous attempts to use laboratory-grown kidneys in laboratory animals have failed because while the transplanted kidneys made urine, they were unable to pass that urine to the animal’s bladder and the kidneys swelled up and failed. Yokoo and his collaborators and colleagues used a stem cell method to make their mini kidneys, as others have in the past.  However, he and his team grew more than just the kidney for the host animal; that also grew a drainage tube, known as a ureter, as well, in addition to a  bladder to collect and store the urine.

Yokoo and others used laboratory rats as the incubators for their growing tissue.  When they connected the new kidney and its tubular systems to the animal’s existing bladder, the system worked.  Urine passed from the transplanted kidney into the transplanted bladder and then into the rat bladder.  The transplant was still working well when they checked eight weeks later.  Then Yokoo and others repeated their procedure in pigs, which are larger mammals than rats and better model systems for human beings.  Fortunately, they achieved the same results.

Although this technology is still years away from clinical trials with human patients, this work provides a paradigm for making organs in the laboratory that will work in sick people.  In the United Kingdom alone, more than 6,000 people are waiting for a kidney.  Because of a shortage of kidney donors, fewer than 3,000 transplants are carried out each year, and more than 350 people die each year waiting for a transplant.  Growing new kidneys using human stem cells could solve this problem.

“To our knowledge, this is the first report showing that the SWPU system may resolve two important problems in the generation of kidneys from stem cells: construction of a urine excretion pathway and continued growth of the newly generated kidney,” Yokoo and others wrote in their paper, which was published in the Proceedings of the National Academy of Sciences, USA, which was communicated to the journal by National Academy of Science member R. Michael Roberts from the University of Missouri-Columbia.

Stem cell expert Prof Chris Mason from University College London, said: “This is an interesting step forward. The science looks strong and they have good data in animals.  But that’s not to say this will work in humans. We are still years off that. It’s very much mechanistic. It moves us closer to understanding how the plumbing might work.  At least with kidneys, we can dialyse patients for a while so there would be time to grow kidneys if that becomes possible.”

Regenerating Heart Muscle with Powdered ECM

Healing the heart after a heart attack is a tough venture. Stem cell treatments have shown definite glimmers to success, but a lack of consistency is a persistent problem. Kick-starting the resident stem cell population in the heart is also a possibility but no single strategy has emerged as a tried and true method to treat a sick heart. Tissue engineering remains an engaging possibility and in the laboratory of Amit Patel at the University of Utah, the possibilities push the boundaries on your imagination.

Patel and his colleagues have been hammering at this problem for decades. The problem is how you replace dead tissue in a beating heart with live tissue that can beat in sync with the rest of the tissue. Unfortunately, you cannot ask the heart to take a vacation to help heal itself. Presently, Patel said that “The doctors say, ‘We’ll give you the beta blocker and the aspirin and the Lipitor and we can just hope to maintain you. But short of them getting worse or getting a heart transplant, there’s [sic] not too many options.”

Patel’s work, however, might change all that. He is presently leading trials on an experimental technology that might repair scarred heart tissue and even arrest or, perhaps, reverse heart failure.

His procedure is in a Phase 1 FDA clinical trial. The trial is designed to mix a powder that consists of a mixture of proteins and molecules isolated from heart muscle with saline or water, inject this mixture into the dead portions of the patient’s heart by means of a catheter, and then wait three to six months to determine if the patient’s heart muscle regenerates.

Dr. Amit Patel puts liquid matrix into a syringe in his lab at University Hospital in Salt Lake City on Wednesday, Sept. 16, 2015. (Photo: Laura Seitz, Deseret News)
Dr. Amit Patel puts liquid matrix into a syringe in his lab at University Hospital in Salt Lake City on Wednesday, Sept. 16, 2015. (Photo: Laura Seitz, Deseret News)

“Heart disease is the most common cause of death in the world, and the most prominent problem is heart failure,” said Tim Henry, the director of cardiology at the Cedars-Sinai Heart Institute. “Effectively, it’s basically one of the biggest problems in the U.S.” Curing the heart with stem cells is, according to Henry, “within our reach,” and Patel, is, to Henry’s thinking, “is clearly one of the most experienced stem cell people in the country”

After a heart attack, the dead regions of the heart form a scar that does not contract, does not conduct electrical impulses, and the rest of the heart has to work around. Reviving the heart scar, shrinking it or reprogramming it to live again has been the dream of stem cell therapy and gene therapy research. However, according to Patel, these venues have not proven to be very good at regenerating dead scar tissue.

Patel, however, noted that “endocardial matrix therapy” would probably be cheaper than stem cell or gene therapy, since it requires an off-the-shelf product that has the advantage of being mass-produced, is easily delivered clinically speaking, and can be easily commercialized and marketed.

This leads to a new question: “What is “extracellular matrix therapy?”

The extracellular matrix is a foundational material upon which cells sit. Extracellular matrix or ECM also provides the glue that attaches cells to each other, layers of cells to each other, and binds tissues together. In Patel’s rendering, ECM consists of everything in our tissues and organs except the cells. If you were to break down the ECM to its parts, you would end up with a concoction of proteins, minerals and a whole cadre of small molecules that can provide a scaffold for cells, nerves and vessels to attach.

To emphasize the importance of the ECM for the heart, Patel said: “A heart without scaffolding is just a bag of cells.” That pretty well nails it.

The ECM also plays a very important signaling role, since it acts as a repository for important signaling molecules that tell cells to grow and develop or divide and heal. The ECM is the milieu in which cells live and grow.

The foundational importance of the ECM gave Patel a revolutionary thought: to heal the heart the matrix has to come first before the cells can follow.

The powder form of heart-specific ECM was developed by scientists at the University of California, San Diego. This group removed the heart muscle from pig hearts, washed away all the cells, and then freeze-dried the remaining ECM into a powder. Using this work as their template, Patel and his team have also devised a protocol to make ECM power from human heart muscle.

When you add water or saline to this ECM powder, it forms a gooey substance called a “hydrogel.” This hydrogel has been called “VentriGel” and it is as flexible as native tissue. Hydrogels are the mainstay of tissue engineering experiments. VentriGel and hydrogels like it can mimic the molecular environment in which cells normally grow and develop. Fortunately, VentriGel has already been shown to successfully reduce scar tissue in the hearts of rats and pigs. To test VentriGel in human patients, Patel and his co-workers can come to the forefront.

Patel recruited a Utah woman who had suffered a heart attack six months ago. This episode reduced her overall heart blood pumping ability from 60 percent (normal) to less than 45 percent (well below normal). Patel and his colleagues made a virtual model of the inside of the patient’s heart to determine where her dead heart muscle resided. Then they marked out 18 different injection sites, and used a catheter to inject the matrix into her heart. The matrix injection procedure took less than two hours.

“This first patient was able to be done awake and safe and she’s already back to work,” Patel said. “She went home the next day.”

Patel plans to treat up to eighteen patients with his experimental procedure. Additionally, cardiologists at the Minneapolis Heart Institute in Minnesota, the only other site approved to test the new technology, performed the procedure on a second patient on Tuesday.

The risks of this procedure are well-known: When hydrogels are directly injected into the heart muscle, they can unintentionally interrupt the electrical conduction of the heart and cause irregular heartbeats. Also, the injected matrix can travel to other parts of the body where it can form a clot that could lead to a stroke. Clots in other parts of the body can also cause the patient’s blood vessels could collapse.

“If you go through all the bad things that could happen, you’d be so depressed, you’d be like, ‘Really? You found somebody to go through this?'” Patel said. “The key is that the team that we have here, and many of my collaborators, we’re all at that same level of healthy enthusiasm mixed with extreme paranoia.”

All patients will be examined three and six months after the procedure out for evidence of muscle regrowth and revived heart function.

“We want to treat this before it ends up leading to permanent damage,” Patel said.

If the trial returns positive results, it will represent another step forward in a long journey to eradicate heart disease. Patel estimates, that if everything goes smoothly, the technology could become approved for clinical use within five to seven years.

Delivery of a Missing Protein Heals Damaged Hearts in Animals

Stanford University School of Medicine scientists have enabled the regeneration of damaged heart tissue in animals by delivering a protein to it by means of a bioengineered collagen patch.

“This finding opens the door to a completely revolutionary treatment,” said Pilar Ruiz-Lozano, PhD, associate professor of pediatrics at Stanford. “There is currently no effective treatment to reverse the scarring in the heart after heart attacks.”

Ruiz-Lozano and her colleagues published their data online in the journal Nature.

During a heart attack, cardiac muscle cells or cardiomyocytes die from a lack of blood flow. Replacing dead cells is vital for the organ to fully recover, but, unfortunately, the adult mammalian heart does not possess a great deal of regenerative ability. Therefore, scar tissue forms instead of heart muscle, and since scar tissue does not contract, it compromises the ability of the heart to function properly.

Heart attacks kill millions of people every year, and the number of heart attacks is predicted to rise precipitously in the next few decades. The number of heart attacks might even triple by 2030. Approximately, 735,000 Americans suffer a heart attack each year, and even though many victims survive the initial injury, the resulting loss of cardiomyocytes can lead to heart failure and even death. “Consequently, most survivors face a long and progressive course of heart failure, with poor quality of life and very high medical costs,” Ruiz-Lozano said. Transplanting healthy muscle cells and stem cells into a damaged heart have been tried, but these trials have mixed results, typically, and have yet to produce consistent success in promoting healing of the heart.

Previous heart regeneration studies in zebrafish have shown that the outer layers of the heart, known as the epicardium, is one of the driving tissues for healing a damaged heart. Ruiz-Lozano said, “We wanted to know what in the epicardium stimulates the myocardium, the muscle of the heart, to regenerate.” Since adult mammalian hearts do not regenerate effectively, Ruiz-Lozano and her co-workers wanted to know whether epicardial substances might stimulate regeneration in mammalian hearts and restore function after a heart attack.

She and her colleagues focused on Fstl1, which is a protein secreted by the epicardium, and acts as a growth factor for cardiomyocytes. Not only did this protein kick-start the proliferation of cardiomyocytes in petri dishes, but Ruiz-Lozano and others found that it was missing from damaged epicardial tissue following heart attacks in humans.

Next, Ruiz-Lozano and her colleagues reintroduced Fstl1 back into the damaged epicardial tissue of mice and pigs that had suffered a heart attack. They embedded a bioengineered patch on to the damaged heart tissue that was imbued with Fstl1. Then they sutured the patch, loaded with Fstl1, to the damaged tissue. These patches were made of natural material known as collagen that had been structurally modified to mimic certain mechanical properties of the epicardium.

Because the patches are made of collagen, they contain no cells, which mean that recipients do not need immunosuppressive drugs to avoid rejection. With time, the collagen material is absorbed into the heart. The elasticity of the material resembles that of the fetal heart, and seems to be one of the keys to providing a hospitable environment for muscle regrowth. New blood vessels regenerated there as well.

Within two to four weeks of receiving the patch, heart muscle cells began to proliferate and the animals progressively recovered heart function. “Many were so sick prior to getting the patch that they would have been candidates for heart transplantation,” Ruiz-Lozano said. The hope is that a similar procedure could eventually be used in human heart-attack patients who suffer severe heart damage.

The work integrated the efforts of multiple labs around the world, including labs at the Sanford-Burnham-Prebys Medical Discovery Institute in San Diego, UC-San Diego, Boston University School of Medicine, Imperial College London and Shanghai Institutes for Biological Sciences.

Stanford has a patent on the patch, and Ruiz-Lozano is chief scientific officer at Epikabio Inc., which has an exclusive option to license this technology.

Cartilage Repair Using Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Embedded in Hyaluronic Acid Hydrogel in a Minipig Model

Cartilage shows lousy regenerative capabilities. Fortunately, it is possible to regenerate cartilage with human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) that have been embedded in a hyaluronic acid (HA) hydrogel composite. In fact, such a combination has shown remarkable results in rat and rabbit models.

In this present study, published in Stem Cells Translational Medicine, Yong-Geun Park and his colleagues from SungKyunKwan University School of Medicine, in Seoul, South Korea sought to confirm the efficacy of this protocol in a in a pig model using three different hUCB-MSC cell lines.

Park and his coworkers generated full-thickness cartilage injuries in the trochlear groove of each knee in 6 minipigs. Three weeks later, an even larger cartilage defect, 5 mm wide by 10 mm deep, was created, followed by an 8-mm-wide and 5-mm-deep boring. In short, the knee cartilages of these minipigs were very messed up.


To these knee cartilages, a mixture (1.5 ml) of hUCB-MSCs (0.5 × 107 cells per milliliter) and 4% HA hydrogel composite were troweled into was then cartilage defects of the right knee. The left knee served as an untreated control. Each cell line was used in two minipigs.

Macroscopic findings of the osteochondral defects of the porcine knees. At 12 weeks postoperatively, the defects of both knees had produced regenerated tissues that were pearly white and firm. These new tissues, which resembled articular cartilage, appeared adherent to the adjacent cartilage and had restored the contour of the femoral condyles (smooth articular surfacewithout depression). The regenerated tissue of the control knee (left knee) looked fibrillated. Grossly, no differencewas seen in the quality of the repaired tissue in the transplanted knee (right knee) among the three groups with different cell lines. (A): Group A. (B): Group B. (C): Group C. Abbreviations: HA, hyaluronic acid; hUCB-MSCs, human umbilical cord blood-derived mesenchymal stem cells.
Macroscopic findings of the osteochondral defects of the porcine knees. At 12 weeks postoperatively, the defects of both
knees had produced regenerated tissues that were pearly white and firm. These new tissues, which resembled articular cartilage, appeared adherent to the adjacent cartilage and had restored the contour of the femoral condyles (smooth articular surface without depression). The regenerated tissue of the control knee (left knee) looked fibrillated. Grossly, no difference was seen in the quality of the repaired tissue in the transplanted knee (right knee) among the three groups with different cell lines. (A): Group A. (B): Group B. (C): Group C. Abbreviations: HA, hyaluronic acid; hUCB-MSCs, human umbilical cord blood-derived mesenchymal stem cells.

12 weeks after surgery, the pigs were sacrificed, and the degree of subsequent cartilage regeneration was evaluated by gross and more detailed microscopic analysis of the knee tissue. The transplanted knee showed superior and more complete joint-specific (hyaline) cartilage regeneration compared with the control knee. The microscopic characteristics of the knee cartilage showed that those animals that received the hUCB-MSCs had greater rates of cell proliferation and cells that differentiated into cartilage-making cells.

Microscopic findings of the regenerating osteochondral defects on porcine articular cartilage (safranin O and fast green staining). At 12 weeks postoperatively, the surface of the repairing tissue in the control knee (left knee) was poorly stained for glycosaminoglycan. In the transplanted knee (right knee), both the regenerated tissue and the adjacent cartilage to which it had become adherent exhibited the normal orthochromatic staining properties with safranin O. (A): Group A. (B): Group B. (C): Group C. Scale bars = 2 mm. Abbreviations: HA, hyaluronic acid; hUCB-MSCs, human umbilical cord blood-derived mesenchymal stem cells.
Microscopic findings of the regenerating osteochondral defects on porcine articular cartilage (safranin O and fast green staining). At 12 weeks postoperatively, the surface of the repairing tissue in the control knee (left knee) was poorly stained for glycosaminoglycan. In the transplanted knee (right knee), both the regenerated tissue and the adjacent cartilage to which it had become adherent exhibited the normal orthochromatic staining properties with safranin O. (A): Group A. (B): Group B. (C): Group C. Scale bars = 2 mm. Abbreviations: HA, hyaluronic acid; hUCB-MSCs, human umbilical cord blood-derived mesenchymal stem cells.

These data show consistent cartilage regeneration using composites of hUCB-MSCs and HA hydrogel in a large animal model. These experiments could be a stepping stone to a human clinical trial in the future that treats osteoarthritis of the knees with hUCB-MSCs embedded in HA hydrogel.

Using Silk to Grow Salivary Glands in the Laboratory

Colloquially, we use the word “spit” to describe saliva, which is secreted by our salivary glands. Saliva is a complex combination of water, salts, proteins, small molecules, and other components that lubricate our throats and mouths to facilitate swallowing, coat our gums and teeth to maintain good gum health and keep tooth decay at bay, and keep our breath fresh. Insufficient salivation production increases tooth decay rates, causes chronic halitosis (bad breath), and can swallowing difficult. Additionally, there are no treatments for poorly-functioning salivary glands, and these glands have poor regenerative capabilities.

Patients who suffer from head/neck cancers and have been treated with radiation suffer from “xerostoma” or dry mouth. Certain medications can also cause dry mouth as can old age. 50% of older Americans suffer from xerostoma.

If that isn’t bad enough, salivary glands are notoriously hard to grow in the laboratory.  So they slow down when we grow old, do not regenerate and grow poorly, in at all, in the laboratory.  Is there any good news about salivary glands?

Make that a yes!  A research team from the University of Texas Health Science Center, led by Chih-Ko Yeh has discovered a process that may lead to the growth of salivary glands in cell culture.  Yeh and his team used purified silk fibers that had many of their contaminants removed to grow salivary stem cells from rat salivary glands.  These cells grew in the laboratory and after several weeks in culture, generated a three-dimensional matrix that covered the silk scaffolds and shared many characteristics with the salivary glands that grow in the mouth.

Yeh underscored the importance of this discovery: “Salivary gland stem cells are some of the most difficult cells to grow in culture and retain their function.”  This work in Yeh’s laboratory have is the first time that salivary gland stem cells have been grown in cell culture while retaining their salivary gland properties.

Yeh continued, “The unique culture system has great potential for future salivary gland research and for the development of new cell-based therapeutics.”

Silk, contrary to what you might think, is an excellent choice for stem cell scaffolding because it is natural, biodegradable, flexible, porous material that provides cells easy access to oxygen and nutrition.  Silk also does not cause inflammation, which is a problem with other types of stem cell scaffolds.

Since there are so few salivary gland stem cells in the human mouth, Yeh and his group plan to continue using the rat model to refine their techniques.  Eventually, Yeh and others would like to use stem cells derived from bone marrow or umbilical cord blood to regenerate salivary glands in human patients.

In fact, Yeh and his coworkers have pioneered protocols for harvesting large numbers of bone marrow stem cells from bone marrow and human umbilical cord blood and growing them in culture.  These stem cells are abundant and can be differentiated into different cell types by means of tissue engineering technologies.

Yeh hopes that by the next decade, human salivary stem cells or tissue engineered artificial salivary gland will be used to initiate salivary gland regeneration in human patients.

This research was published in Tissue Engineering part A 2015; 21(9-10).

Bioabsorbable Cardiac Matrix Fails in Phase 1 Clinical Trial

A clinical trial known as Preservation 1 examined the use of Bioabsorbable Cardiac Matrix in heart attack patients. Preservation 1 was a double-blinded, placebo-controlled study that examined heart attack patients who had been treated with PCI (percutaneous coronary intervention), which is also known as angioplasty with stent placement.

This clinical trial compared patients who had the bioabsorbable cardiac matrix placed over the dead part of the heart with those who had been treated with sterile saline (placebo). Because it was a phase 1 clinical trial, it tested safety issues rather than efficacy issues.

The bioabsorbable cardiac matrix is initially administered as a liquid that is injected into the heart. Upon contact with damaged cardiac tissue, it undergoes a transition from liquid to gel that provides mechanical support and allows the damaged tissue to heal and form a more compact, tighter scar. A smaller scar should result in improved long-term cardiac function. The gel also resorbs naturally and is excreted from the body within six weeks of injection. The matrix might also be able to support stem cells that migrate to the heart after the heart attack. Therefore, the hope for this trial was that patients who received the bioabsorbable cardiac matrix might show less remodeling or enlargement of the heart after their heart attacks. Patients were given either the placebo of the bioabsorbable cardiac matrix 2-5 days after receiving PCI.

When studied in laboratory rodents, the matrix was administered up to 7 days post-heart attack and it improved survival, prevented the dilation of the left ventricular end systolic and diastolic volume, prevented fractional shortening deterioration and improved mitral regurgitation. Treatment also minimized the systolic wall thinning, all of which are findings consistent with the prevention of progressive expansion of the infarcted area of the heart.

In the Preservation 1 trial, patients were examined six months after treatment, and their left ventricular end diastolic volume index (LVEDVI) was measured. The LVEDVI goes up if the heart is undergoing remodeling. Also patients were given a standard questionnaire (Kansas City Cardiomyopathy Questionnaire) and a six-minute walk test. Adverse reactions that were measured were time until the patients died from heart-related issues, heart-related events, hospitalizations, and time until the first heart-related hospitalization.

Unfortunately, when it came to safety and efficacy, the bioabsorbable cardiac matrix was no better than the placebo. This product, which was called BL-1040, was licensed to a company called Bellerophon in 2009. Prior to this partnership with Bellerophon, BioLineRx invested some $10 million in the development BL-1040. It is a shame that it failed in this trial.

Biolinerx is still eager to test their product as a way to mobilize cells from bone marrow, induce the death of cancer cells, and other potential applications.  While the theory behind this product seems sound, the heart after a heart attack needs more than just a temporary structural support; it needs cells and new vasculature.  If the bioabsorbable cardiac matrix could be laced with stem  cells and the matrix could not only contribute to stem cell survival, but also stem cell efficacy, then this product might really help a damaged heart heal.

Stem Cells Embedded in a Fibrin Patch Help Hearts Recover After a Heart Attack

If a patient has a heart failure, there is little you can do for them. Medications can take some of the stress off the failing heart, and in extreme cases, a heart transplant is warranted. However, organ transplants are hampered by both the limited number of organ donors and the potential for the patient’s body to reject the new heart.

A new study from the journal STEM CELLS Translational Medicine has shown that heart tissue can be regenerated if engineered patches made up of a mixture of fibrin and mesenchymal stem cells (MSCs) derived from human umbilical cord blood are applied to the heart.

Previous studies show the potential of MSCs to repair damage generated by a heart attack. In these clinical studies, the MSCs were delivered through injections into the heart muscle or intravenously. “While feasible and safe, the treatments exhibited only modest benefits,” said Antoni Bayes-Genis, M.D., Ph.D., member of the ICREC (Heart Failure and Cardiac Regeneration) Research Program, Germans Trias i Pujol Health Science Research Institute (IGTP) and professor at Universitat Auto`noma de Barcelona. Dr. Bayes-Genis is a lead investigator on this study.

“The survival rate of the implanted stem cells was generally low and about 90 percent of them either died or migrated away from the implantation site, generally to the liver,” added the study’s first author, Santiago Roura, Ph.D., also a member of the ICREC Research Program and IGTP. “These limited effects are probably due to the adverse mechanical stress and hypoxic conditions present in the myocardium after the heart attack.”

Now could a better way to deliver the MSCs to the injured site yield more efficient results? Synthetic scaffolds (or patches) in which the cells are embedded in matrices constructed of biological and/or synthetic materials and supplemented with growth or differentiation factors can generate so-called “bioimplants.” Bioimplants are a promising way to potentially apply stem cells to the heart in a way that will allow them to survive, grow and thrive. Unfortunately, none of the current materials being tested for heart patches, whether synthetic or natural has been shown to provide optimal properties for cardiac tissue repair.

Dr. Bayes-Genis and his colleagues examined how a fibrin patch filled with human umbilical cord blood-derived MSCs might serve to repair a damaged heart. Fibrin is widely used in medical applications, since it can act as a bio-compatible glue that holds cells in place and stimulating the production of new blood vessels (angiogenesis). Bayes-Genis and others hypothesized that fibrin scaffolds might offer a nurturing environment for the growth and proliferation of MSCs at the site of the heart injury. There, the cells could induce the repair of damaged heart tissue.

Bayes-Genis and coworkers mixed MSCs and fibrin to form the patches that were then applied to the hearts of mice that had undergone heart attacks. Three weeks later, they compared the recovery of these animals to a control group of mice that were treated with fibrin alone without embedded stem cells, and a third group that received no treatment at all. The results showed that the patches adhered well to the hearts and the MSCs grew and differentiated. The patch cells also participated in the formation of new, functional blood vessels that connected the patch to both the heart tissue directly beneath it and the mouse’s endogenous circulatory system, too.

“As a result, the heart function in this group of mice was better than that of the animals in either of the other control groups,” Dr. Bayes-Genis said. “Thus, this study provides promising findings for the use of umbilical cord-blood MSCs and fibrin patches in cardiac repair.”

“This is an interesting study that suggests a news strategy for using stem cells to repair injured heart tissue, without the drawbacks that cell injections have shown,” said Anthony Atala, M.D., Editor-in-Chief of STEM CELLS Translational Medicine and Director of the Wake Forest Institute for Regenerative Medicine.

Bioengineered Nanofiber Patch to Treat Heart Failure

Stem cells have the capacity to heal damaged tissues and replace death cells. However, harvesting and employing the right stem cell for the right job, at the right dose, and under the right conditions has proven to be a difficult puzzle to solve.

In particular, the damaged heart has proven rather difficult to heal with stem cells. Many different clinical trials have administered stem cells by direct injection, intracoronary delivery in coronary vessels, or injection into the surface of the heart. These studies have examined the efficacy of stem cells from fat, bone marrow, the heart itself, and other sources. The upshot of these studies is that some strategies work and others do not, but even those that work only work modestly well.

The biggest obstacle is overcoming the hostile environment into which stem cells are introduced when they are administered into the heart after a heart attack. The post-heart attack heart suffers from lots of inflammation, low oxygen concentrations, and the pervasive presence of dangerous molecules. Several laboratories have discovered that preconditioning stem cells by growing them in low-oxygen culture conditions can increase their survival in the post-heart attack heart as can genetically engineering cells to resist increased levels of cell stress. Now, a research team from Ohio State University has designed a different strategy to beat the hostility of the post-heart attack heart.

After a heart attack, oxygen-deprived tissues die and various chemical messengers instruct damaged cells to die. However, data from a host of clinical trials strongly suggests that this dangerous time is the best time to introduce stem cells into the heart. Thus, this window of opportunity is the “best of times and the worst of times” for cell therapy.

As it turns out, about 30% of all mammalian protein-encoding genes are regulated by small molecules called microRNAs (miRNAs). MiRNAs are single-stranded RNA molecules approximately 22 nucleotides in length that bind to messenger RNAs and regulate their translation into protein or half-life. Research has shown that miRNAs have substantial potential as a therapeutic target for the treatment of many diseases, including cardiovascular disease. A good deal of research in laboratory animals and in cultured heart cells that altered expression of miRNAs such as miR-1, miR-133, miR-21 and miR-208 contribute to the development of heart disease. The laboratory of Mahmood Khan, a scientist at the Davis Heart and Lung Research Institute at The Ohio State University Wexner Medical Center, has focused on miR-133a which seems to play a role in slowing fibrosis and cardiac remodeling. Importantly, levels of miR-133a are reduced in the heart tissues of patients who have suffered a heart attack.

Khan and his group predicted that increasing the levels of miR-133a in stem cells as they are cultured might preprogram the cells to survive in the hostile environment of the post-heart attack heart.

Khan’s team began by bioengineering a molecule to induce mesenchymal stem cells (MSCs) to produce miRNA-133a. When transplanted into an animal model of cardiac ischemia, the pre-treated MSCs showed improved survival over non-treated MSCs. These pre-treated MSCs also did a better job at decreasing the global damage to the heart, and increasing the thickness of the left ventricle, the main pumping chamber of the heart.

“We found that the pre-treated MSCs did a better job at decreasing the global damage to the heart, along with improvement in the left-ventricular wall thickness compared to the untreated MSCs,” said Dr. Angelos. “MSCs are a commonly used cell type in current heart failure studies, so our findings are definitely relevant to that work.”

The results of these experiments were published in the March issue of the Journal of Cardiovascular Pharmacology.

While trying to increase cell survival, Khan and his colleagues also addressed the problems surviving stem cells face in the heart – how to help them function along existing heart tissue without getting in the way or fouling things up.

To date, most stem cells are grown in flat culture plates and either injected directly into the heart muscle (typically on the periphery of scar tissue), or infused into the heart via an artery. While most of these stem cells either die or diffuse throughout the body, successfully transplanted stem cells sometimes inadvertently disrupt heart function.

“The heart is a constantly moving, connected matrix of muscle fibers working together to make the heart pump in sync,” said Dr. Angelo’s, an emergency medicine researcher and collaborator of Dr Khan. “Transplanted stem cells may not align with native tissue, potentially disrupting or attenuating signals that keep a steady heartbeat. There’s evidence that this could contribute to arrhythmias.”

To create a more secure environment that allows implanted stem cells successfully engraft into the heart, Drs. Khan and Angelos have used a biodegradeable nanofiber “patch” seeded with human inducible pluripotent stem cells derived cardiomyocytes (hiPSC-CMs). Khan and Angelos chose hiPSC-CMs because these cells are patient derived and can be used to model the heart disease of patients and for autologous stem cell transplantation in patients with failing hearts.

Both aligned nanofiber patch and standard culture plate were seeded with hiPSC-CMs. Both sets of cultures heart muscle cells were compared for calcium signaling (a measure of proper heart muscle function) and synchronous beating. Within two weeks, both stem cell cultures were spontaneously beating like a miniature heart, but the linear grain of the nanofiber formed an aligned pattern of cells that looked and functioned like a healthy heart tissue.

“The cardiomyocytes cultured on a flat plate are scattered and disorganized. Cardiomyocytes grown on the nanofiber scaffolding look more like healthy heart cells, beat more strongly and in greater synchronicity than cells from the flat plate,” said Dr. Khan. “Next, we hope to use what we’ve learned from this study to develop a thicker, multi-layer patch that could help restore thin and weakened heart walls.”

Drs. Khan and Angelos see great potential future clinical applications for their nanofiber bandage. This treatment could potentially bandage the damaged heart muscle of heart patients with the nanofiber cardiac patch. Also, it is possible that they could someday combine the microRNA pre-treatment technique and the patch to give stem cells a survival boost along with a protective structure to improve outcomes.

Khan, his co-workers and his collaborators published this work on May 19 in PLoS ONE.

New Tissue Engineering Technique Could Lead to Growing Larger Organs in the Laboratory

Tissue engineers from the Universities of Liverpool and Bristol have invented a novel tissue “scaffold” technology that might one day enable the growth large organs in the laboratory.

According to data generated by these experiments, it is possible to combine cells with a special scaffold to produce living tissues in the laboratory. Hopefully, such organs can then be implanted into patients who need to have a diseased body part replaced. To this point, growing large organs in the laboratory has been impossible because growing larger structures in the laboratory limits the delivery of oxygen supply to the cells in the center of the organ. Therefore, growing tissues in the laboratory has been restricted to small structures that are readily served by the diffusion of oxygen.

In the experiments conducted by the University of Liverpool and Bristol teams, cartilage tissue engineering was employed as a model system for testing strategies for overcoming the oxygen limitation problem.

They manufacture a new class of artificial membrane binding proteins that attached to stem cells. Then they attached to these cell surface proteins the oxygen-carrying protein, myoglobin, before they used the cells to engineer cartilage. Since myoglobin is an oxygen-storage molecule, it will bind oxygen and provide a reservoir of oxygen for cells that cells can access when the oxygen in the scaffold drops to dangerously low levels.

Professor Anthony Hollander, Head of the University of Liverpool’s Institute of Integrative Biology, said: “We have already shown that stem cells can help create parts of the body that can be successfully transplanted into patients, but we have now found a way of making their success even better. Growing large organs remains a huge challenge but with this technology we have overcome one of the major hurdles. Creating larger pieces of cartilage gives us a possible way of repairing some of the worst damage to human joint tissue, such as the debilitating changes seen in hip or knee osteoarthritis or the severe injuries caused by major trauma, for example in road traffic accidents or war injuries.”

These results could expand the possibilities in tissue engineering, not only in cartilage, but also for other tissues such as cardiac muscle or bone. This new methodology in which a normal protein is converted into a membrane binding protein to which helpful molecules can be attached, is likely to pave the way for the development of a wide range of new biotechnologies.

Dr Adam Perriman, from the University of Bristol, added: “From our preliminary experiments, we found that we could produce these artificial membrane binding proteins and paint the cells without affecting their biological function. However, we were surprised to discover that we could deliver the necessary quantity to the cells to supplement their oxygen requirements. It’s like supplying each cell with its own scuba tank, which it can use to breathe from when there is not enough oxygen in the local environment.”

Previous work by Hollander’s group includes the development of a method of creating cartilage cells from stem cells. This method helped make the first successful transplant of a tissue-engineered trachea, which utilized the patient’s own stem cells, possible.

This work appeared in the paper, “Artificial membrane-binding proteins stimulate oxygenation of stem cells during engineering of large cartilage tissue,” which was published in Nature Communications.

Rebuilding Bladders with Cell-Free Materials

Ying-jian Zhud and Mu-jun Luan from the Shanghai Jiao Tong University in Shanghai, China teamed up to examine a new way to regenerate the bladder.

Several different synthetic and natural biomaterials have been pretty widely used in tissue regeneration experiments, particularly in the regeneration of the urinary bladder. The vast majority of this work has been done in rat model systems, which are fairly good animals to model bladder pathology and regeneration.

To date, the attempted reconstructive procedures don’t seem to work all that well, and this is due to the lack of appropriate scaffolding upon which cells can attach, grow and spread to form the new bladder tissue. Any scaffolding material for the bladder has to provide a waterproof barrier and it has to be able to support several different cell types. While this might not sound difficult on paper, it is in fact rather difficult. Some biomaterials might be well tolerated by the body, but cannot be fashioned into the shape of the organ. Others might support the growth of cells quite well, but are not tolerated by the body.

Zhud and Luan addressed these issues by turning to two different compounds that would compose a two-layered structure. Such a two-layered structure would support the cell types of the bladder. The outside layer was composed of silk fibroin, which is very moldable and usually well tolerated by cells. The inner layer consisted of a natural, acellular matrix (or BAMG for bladder acellular matrix graft). They used this two-layered structure to regenerate an injured bladder in rats.

First of all, it was clear that this material was relatively easy to make and it also could be nicely molded and sewn into the existing bladder. Tissue stains showed something even more interesting: the bilayer scaffold promoted the growth and recruitment of smooth muscles, blood vessels, and even nerves in a time-dependent manner. So by 12 weeks after implantation, bladders reconstructed with the bilayered matrix displayed superior structural and functional properties without significant local tissue responses or systemic toxicity.

Thus, the silk/BAMG scaffold could potentially be a promising scaffold for bladder regeneration. It shows good tissue compatibility, and allows the growth of cells on it. More work is required to take this to the next step, and the scaffold will undoubtedly undergo some changes. But this work represents a terrific start to what might be a superior scaffold for bladder regeneration.

Rat forelimbs grown in the lab

The moving pictures of American soldiers who lost limbs while serving their country come across our computer screens with some regularity. However, while we celebrate the courage of these young men and women, we should also be amazed at the technological advances that provide artificial limbs for these soldiers. What if, we could grow replacement limbs in culture? Is this science fiction? Maybe not.

Biolimb from Ott lab
Biolimb from Ott lab

The photo above comes from work done in the laboratory of Harald Ott who is at the Massachusetts General Hospital in Boston has succeeded in growing rodent forelimbs in the laboratory. “We’re focusing on the forearm and hand to use it as a model system and proof of principle,” said Ott. “But the techniques would apply equally to legs, arms and other extremities.”

“This is science fiction coming to life,” says Daniel Weiss at the University of Vermont College of Medicine in Burlington, who works on lung regeneration. “It’s a very exciting development, but the challenge will be to create a functioning limb.”

Modern amputees are often fitted with prosthetic limbs that have an excellent cosmetic look, but these artificial limbs don’t function as well as real limbs. Bionic replacement limbs that work well are now being made, but they look quite unnatural. Hand transplants have also been successful, but these surgeries are extremely expensive, and the recipient needs lifelong immunosuppressive drugs to prevent their body rejecting the transplanted hand.

Tissue engineered “biolimbs” would get round many of these obstacles as it only contains cells from the recipient and would, therefore, avoid the need for immunosuppression. Biolimbs would also look and behave naturally.

“This is the first attempt to make a biolimb, and I’m not aware of any other technology able to generate a composite tissue of this complexity,” says Ott.

To grow rat forelimbs in the laboratory, the so-called “decel/recel” technique was used. This same technique was previously been used to build hearts, lungs and kidneys in the lab. In fact, simpler organs such as windpipes and voice box tissue have been built and transplanted into people with varying levels of success, but not without controversy.

Decel stands for decellularization is the first step. In the decel step, organs from dead donors are treated with detergents that strips the soft tissue and leaves just the “scaffold” of the organ, which consists mainly from the inert protein collagen. This retains all the intricate architecture of the original organ. In the case of the rat forearm, these collagen structures include blood vessels, tendons, muscles and bones.

The second step, the recel step, recellularizes the flesh of the organ by seeding the scaffold with the relevant cells extracted from the recipient. This scaffold is then nourished in a bioreactor, which enables the new tissue to grow and colonize the scaffold. Because none of the donor’s soft tissue remains, this bioengineered limb, or biolimb, will not be recognized as foreign and rejected by the recipient’s immune system.

As tissue engineered organs go, the forearm is much more difficult to grow that a windpipe. It has a far greater number of cell types that need to be grown. Ott began by suspending the decellularized forelimb in a bioreactor, and then plumbing the collagen artery into an artificial circulatory system to provide nutrients, oxygen and electrical stimulation to the limb. Next, Ott and his colleagues injected human endothelial cells into the collagen structures of blood vessels to recolonize the surfaces of blood vessels. This was important, because, according to Ott, this made the blood vessels more robust and prevented them from rupturing as fluids circulated through them.

Next, he injected a mixture of cells from mice that included myoblasts or muscle forming cells that would grow into muscle in the cavities of the scaffold normally occupied by muscle. In two to three weeks, the blood vessels and muscles had been rebuilt. Ott then finished off the limb by coating the forelimbs with skin grafts.

But would the limb’s muscles work? In order to work, the muscles must be connected to motor nerves from the central nervous system. To try this out, Ott’s team used electrical pulses to activate the muscles and found that the rat’s paw could clench and unclench. This experiment “showed we could flex and extend the hand,” says Ott. They also attached the biolimbs to anaesthetized healthy rats and saw that blood from the rat circulated in the new limb. However, they didn’t test for muscle movement or rejection.

While they have decellularized around 100 rat forelimbs, recellularizing at least half of them, there is still a great deal of work to do, said Ott. First they need to seed the limb with bone, cartilage and other cells to see whether these structures can be grown in the biolimb. Then they must demonstrate that a nervous system will develop in these cells. Results of hand transplants have shown the re-enervation occurs by means of the recipient’s nerve tissue growing into the transplanted hand and penetrating it. These growing nerves then make connections with the appropriate muscles. Thus, Ott believes that this would enable the recipients of a transplanted biolimb to control of their new organ. However, whether this also works in regenerated limbs remains to be seen.

Ott and his colleagues have also shown that forearms from nonhuman primates can be successfully decellularized. His team has begun recolonizing the primate scaffolds with human cells that line blood vessels, which is the first step towards human-scale biolimb development. They have also started experiments using human myoblasts in rats instead of the mouse myoblasts. Considerable work is needed to perfect this technology and it will be at least a decade before the first biolimbs are ready for human testing, says Ott, which is probably an optimistic estimate.

Nonhuman primate limb
Nonhuman primate limb

“It’s a notable step forward, and based on sound science, but there are some technical challenges that Harald’s group has to tackle,” says Steve Badylak of the University of Pittsburgh in Pennsylvania, who has used grafts built on scaffolds made from pig muscle to rebuild damaged leg muscles in 13 people. “Of these, the circulation is probably the biggest challenge, and making sure even the tiniest capillaries are successfully lined with endothelial cells so that they don’t collapse and cause clots,” he says. “But this is really an engineering approach, taking known fundamental principles of biology and applying them as an engineer would.”

Others are more critical. “For a complex organ like the hand, there are so many tissues and compartments that this definitely will not be a feasible protocol,” says Oskar Aszmann of the Medical University of Vienna in Austria, inventor of a bionic hand that people can control through their own thoughts. “Also, the hand must be innervated by thousands of nerves to have meaningful function, and that is at this point an insurmountable problem. So although this is a worthy endeavor, it must at this stage remain in the academic arena, not as a clinical scenario.”

In humans, Ott envisages organ donation schemes being extended to include transplantation of biolimbs. Cells for regenerating blood vessels could come from minor vessels supplied by the recipient, while muscle cells could come from biopsies from large muscles, such as in the thigh. “If you took about 5 grams, the size of a finger, you could grow it into human skeletal myoblasts,” he says.

With 1.5 million amputees in the US alone, this regeneration work is important, says Ott. “At present, if you lose an arm, a leg or soft tissue as part of cancer treatment or burns, you have very limited options.”

Artifical Blood Vessels Made From Thermoplastic Polyurethane Polymers

Wherever we find some of the worse medical events – heart attacks, strokes, pulmonary embolisms, we find blocked blood vessels. Obstructed blood vessels are a lurking time bomb in our bodies and they usually have to be replaced. Blood vessel replacement requires cutting another blood vessel from another part of the body or the implantation of artificial vascular prostheses.

A new option might emerge in the future, however. Vienna University of Technology, in collaboration with the Vienna Medical University developed artificial blood vessels that were fabricated from specialized elastomer material that have excellent mechanical properties. After implantation, these artificial blood vessels are dissolved and replaced by the body’s own blood vessels. At the end of the healing process, natural, fully functional blood vessels are once again in place. The technique works quite well in tissue cultures systems, but now it has been shown to successfully regenerate blood vessels in laboratory animals, specifically rats.

Atherosclerotic vascular disorders, in which blood vessels are obstructed by cholesterol-filled plaques, are one of the most common causes of death in industrialized countries. Typically, patients are treated with a bypass operation, and for such procedures, blood vessels are extirpated from another part of the patient’s body and used to replace the damaged vessel. This creates a new wound and a new area of the body with less than optimal blood supply that must heal. Also, the transplanted vessel rarely has the properties necessary to thrive in its new location.

This new strategy to replace diseased blood vessels is the result of a fruitful collaboration between Vienna University of Technology (or TU Wien, which is short for Technische Universität Wien) and the Medical University of Vienna. Hopefully the success of this research will cause artificially manufactured vessels to be used more frequently in future.

To make an artificial blood vessel, the most important thing is to start with the right material. The material must be compatible with body tissue, and pliable enough to be formed into a small diameter tube that is not easily blocked by blood clots.

Extensive work at TU Wien has resulted in the development of new polymers. “These are so-called thermoplastic polyurethanes,” explains Robert Liska from the Institute of Applied Synthetic Chemistry of TU Wien.  “By selecting very specific molecular building blocks we have succeeded in synthesizing a polymer with the desired properties.”

In order to generate artificial blood vessels from their thermoplastic polyurethanes, TU Wien materials scientists spun polymer solutions in an electrical field. This allowed them to form very fine threads and that could be wound into a spool. “The wall of these artificial blood vessels is very similar to that of natural ones,” says Heinz Schima of the Medical University of Vienna. The thermoplastic polyurethanes form a polymer fabric that is slightly porous and allows a small amount of blood to leak through it. This also enriches the blood vessel wall with growth factors, which encourages the migration of endothelial progenitor cells. Martina Marchetti-Deschmann at TU Wien studied the interaction between the thermoplastic polyurethane material and blood by using spatially resolved mass spectrometry.

This new technology has already proven to successfully form functional blood vessels in rats. “The rats’ blood vessels were examined six months after insertion of the vascular prostheses,” says Helga Bergmeister of MedUni Vienna. “We did not find any aneurysms, thromboses or inflammation. Endogenous cells had colonized the vascular prostheses and turned the artificial constructs into natural body tissue.” In fact, the body’s own blood vessel-forming tissues re-grew significantly faster than expected, which shortened the degradation period of the plastic tubes and their replacement with the body’s own endothelial cells. TU Wein and the Medical University of Vienna are making further adaptations to the material.

A few more preclinical trials are necessary before the artificial blood vessels can be used in human clinical trials. However, based on the results so far, the research team is very confident that the new method will prove itself for use in humans in a few years’ time.

This project was recently awarded PRIZE prototype funding from Austria Wirtschaftsservice (AWS).