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.