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.

Elabela, A New Human Embryonic Stem Cell Growth Factor


When embryonic stem cell lines are made, they are traditionally grown on a layer of “feeder cells” that secrete growth factors that keep the embryonic stem cells (ESCs) from differentiating and drive them to grow. These feeder cells are usually irradiated mouse fibroblasts that coat the culture dish, but do not divide. Mouse ESCs can be grown without feeder cells if the growth factor LIF is provided in the medium. LIF, however, is not the growth factor required by human ESCs, and therefore, designing culture media for human ESCs to help them grow without feeder cells has proven more difficult.

Having said that, several laboratories have designed media that can be used to derive human embryonic stem cells without feeder cells. Such a procedure is very important if such cells are to be used for therapeutic purposes, since animal cells can harbor difficult to detect viruses and unusual sugars on their cell surfaces that can also be transferred to human ESCs in culture. These unusual sugars can elicit a strong immune response against them, and for this reason, ESCs must be cultivated or derived under cell-free conditions. However, to design good cell-free culture media, we must know more about the growth factors required by ESCs.

To that end, Bruno Reversade from The Institute of Molecular and Cell Biology in Singapore and others have identified a new growth factor that human ESCs secrete themselves. This protein, ELABELA (ELA), was first identified as a signal for heart development. However, Reversade’s laboratory has discovered that ELA is also abundantly secreted by human ESCs and is required for human ESCs to maintain their ability to self-renew.

Reversade and others deleted the ELA gene with the CRISPR/Cas9 system, and they also knocked the expression of this gene down in other cells with small interfering RNAs. Alternatively, they also incubated human ESCs with antibodies against ELA, which neutralized ELA and prevented it from binding to the cell surface. However Ela was inhibited, the results were the same; reduced ESC growth, increased amounts of cell death, and loss of pluripotency.

How does ELA signal to cells to grow? Global signaling studies of growing human ESCs showed that ELA activates the PI3K/AKT/mTORC1 signaling pathway, which has been show in other work to be required for cell survival. By activating this pathway, ELA drives human ESCs through the cell-cycle progression, activates protein synthesis, and inhibits stress-induced apoptosis.

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Interestingly, INSULIN and ELA have partially overlapping functions in human ESC culture medium, but only ELA seems to prime human ESCs toward the endoderm lineage. In the heart, ELA binds to the Apelin receptor APLNR. This receptor, however, is not expressed in human ESCs, which suggests that another receptor, whose identity remains unknown at the moment, binds ELA in human ESCs.

Thus ELA seems to act through an alternate cell-surface receptor, is an endogenous secreted growth factor in human

This paper was published in the journal Cell Stem Cell.

Update on First Induced Pluripotent Stem Cell Clinical Trial


Masayo Takahashi, an ophthalmologist at the RIKEN Center for Developmental Biology (CDB) in Kobe, Japan, has pioneered the use of induced pluripotent stem cells (iPSCs) to treat patients with degenerative retinal diseases.

Takahashi isolated skin cells from her patients, and then had them reprogrammed into iPSCs in the laboratory through a combination of genetic engineering and cell culture techniques. These iPSCs have many similarities with embryonic stem cells, including pluripotency, which is the potential to differentiate into any adult cell type.

Once induced pluripotent stem cell lines were established from her patient’s skin cells, they had their genomes sequenced for safety purposes, and then differentiated into retinal pigmented epithelial (RPE) cells. RPE cells lie beneath the neural retina and support the photoreceptors that respond to light. When the RPE cells die off, the photoreceptors also begin to die.

Takahashi watched the transplantation of the RPE cells that she had grown in the laboratory into the back of a woman’s damaged retina. This transplant would constitute the first test of the therapeutic potential of iPSCs in people. Takahashi described the transplant as “like a sacred hour.”

Takahashi has collaborated with Shinya Yamanaka, the discoverer of iPSC technology. She devised ways to convert the iPS cells into sheets of RPE cells. She then tested the resulting cells in mice and monkeys, jumped the various regulatory loops, recruited patients for her clinical trial, and practiced growing cells from those patients. Finally, she was ready to try the transplants in people with a common condition called age-related macular degeneration, in which wayward blood vessels destroy photoreceptors and vision. The transplants are meant to cover the retina, patch up the epithelial layer and support the remaining photoreceptors. Watching the procedure, “I could feel the tension of the surgeon,” Takahashi said.

This transplant surgery occurred approximately a year ago. Some new data on this patient is available.

As of 6 months after the transplant, the procedure appears to be safe. The one-year safety report should appear soon. Prior to the transplant, the patient was a series of 18 anti-vascular endothelial growth factor (anti-VEGF) ocular injections for both eyes to cope with the constant recurrence of the disease. However, data presented by Dr. Takahashi showed that the patient had subretinal fibrotic tissue removed during the transplant surgery in order to make room for the RPE cells. Once the RPE cells were implanted, the patient experienced no recurrence of neovascularization at the 6-month point. This is significant because she has not had any other anti-VEGF injections since the transplant. Her visual acuity was stabilized and there have been no safety related concerns to date.

I must grant that this is only one patient, but so far, these results look, at least hopeful. Hopefully other patients will be treated in this trial, and hopefully, they will experience the same success that the first patient is enjoying. We also hope and pray that the first patient will continue to experience relief from her retinal degeneration.

As to the treatment of the second patient of this trial, Takahashi has hit a snag. Some mutations were detected in the iPS cell-derived RPE cells prepared for the second patient. No one knows if these mutations make these cells dangerous to implant. Regulatory guidelines, at this point, are also no help. Apparently, the cells have three single-nucleotide change and three copy-number changes that are present in the RPE cells that were not detectable in the patient’s original skin fibroblasts. The copy-number changes were, in all cases, single-gene deletions. One of the single-nucleotide changes is listed in a database of cancer somatic mutations, but only linked to a single cancer. Further evaluation of these mutations shows that they were not in “driver genes for tumor formation,” according to Dr. Takahashi.

Tumorigenicity tests in laboratory animals has established that the RPE cells are safe. Remember that the presence of a mutation does not necessarily mean that these RPE cells can be tumorigenic.

However, Takahashi has still decided to not transplant these cells into the second patients. Part of the reason is caution, but the other reason is compliance with new Japanese law on Regenerative Medicine, which became effective after iPS trial was begun. This law, however, does not specify how safe a cell line has to be before it can be transplanted into a patient.

RIKEN’s decision to halt the trial is probably a good idea. After all, this is the first trial with iPSCs and it is important to get it right. Even though the RPE cells were widely thought to be safe to use, Takahashi decided not to implant another patient with RPEs derived from their own cells. Instead, they decided to use RPEs made from donated iPSC lines. Therefore, Takahashi is in discussions government officials to determine how this change of focus for the trial affects their compliance with Japanese law.

Frankly, this might be a very savvy move on Takahashi’s part. As Peter Karagiannis, a spokesperson for the Center for iPS Cell Research and Application, noted: “As of now, autologous would not be a feasible way of providing wide-level clinical therapy. At the experimental level it’s fine, but if it’s going to be mass-produced or industrialized, it has to be allogeneic.”

Therefore, the RIKEN institute is moving forward with allogeneic iPSC-derived RPEs. RIKEN will work in collaboration with the Center for iPS Cell Research and Application (CiRA) in Kyoto, Japan, which has several well characterized, partially-matched lines whose safety profiles have been established by strict, rigorous safety testing methods. However, immunological rejection remains a concern, even if these cells are transplanted into an isolated tissue like the eye where to immune system typically is not allowed. The simple fact is that no one knows if the cells will be rejected until they are used in the trial.

An additional concern is that CiRA has not typed its cells for minor histocompatibility antigens, which can cause T cell–mediated transplant rejection.

Nevertheless, Takahashi and her team deserve a good deal of credit for their work and vigilance.

MSC Transplantation Reduces Bone Loss via Epigenetic Regulation of Notch Signaling in Lupus


Mesenchymal stem cells from bone marrow, fat, and other tissues have been used in many clinical trials, experiments, and treatment regimens. While these cells are not magic bullets, they do have the ability to suppress unwanted inflammation, differentiate into bone, cartilage, tendon, smooth muscle, and fat, and can release a variety of healing molecules that help organs from hearts to kidneys heal themselves.

Mesenchymal stem cell transplantation (MSCT) is the main means by which mesenchymal stem cells are delivered to patients for therapeutic purposes. However, the precise mechanisms that underlie the success of these cells are not fully understood. In a paper by from the University Of Pennsylvania School Of Dental Medicine published in the journal Cell Metabolism, MSCT were able to re-establish the bone marrow function in MRL/lpr mice. The MRL/lpr mouse is a genetic model of a generalized autoimmune disease sharing many features and organ pathology with systemic lupus erythematosus (SLE). Such mice show bone loss and poor bone deposition, a condition known as “osteopenia.” Because mesenchymal stem cells are usually the cells in bone marrow that differentiate into osteoblasts (which make bone) a condition like osteopenia results from defective mesenchymal stem cell function.

In this paper, Shi and his coworkers and collaborators showed that the lack of the Fas protein in the mesenchymal stem cells from MRL/lpr mice prevents them from releasing a regulatory molecule called “miR-29b.” This regulatory molecule, mir-29b, is a small RNA molecule known as a microRNA. MicroRNAs regulate the expression of other genes, and the failure to release miR-29b increases the intracellular levels of miR-29b. This build-up in the levels of miR-29b causes the downregulation of an enzyme called “DNA methyltransferase 1” or Dnmt1. This is not surprising, since this is precisely what microRNAs do – they regulate genes. Dnmt1 attaches methyl groups (CH3 molecules) to the promoter or control regions of genes.

Decrease in the levels of Dnmt1 causes hypomethylation of the Notch1 promoter. When promoters are heavily methylated, genes are poorly expressed. When very methyl groups are attached to the promoters, then the gene has a greater chance of being highly expressed. Robust expression of the Notch1 genes activates Notch signaling. Increased Notch signaling leads to impaired bone production, since differentiation into bone-making cells requires mesenchymal stem cells to down-regulate Notch signaling.

When normal mesenchymal stem cells are transplanted into the bone marrow of MRL/lpr mice, they release small vesicles called exosomes that transfer the Fas protein to recipient MRL/lpr bone marrow mesenchymal stem cells. The presence of the Fas protein reduces intracellular levels of miR-29b, and this increases Dnmt1-mediated methylation of the Notch1 promoter. This decreases the expression of Notch1 and improves MRL/lpr BMMSC function.

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These findings elucidate the means by which MSCT rescues MRL/lpr BMMSC function. Since MRL/lpr mice are a model system for lupus, it suggests that donor mesenchymal stem cell transplantation into lupus patients provides Fas protein to the defective, native mesenchymal stem cells, thereby regulating the miR-29b/Dnmt1/Notch epigenetic cascade that increases differentiation of mesenchymal stem cells into osteoblasts and bone deposition rates.

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.

Some Types of Obesity Might be Caused by a Faulty Immune System


When we think of our Immune systems, we normally entertain visions of white blood cells that fight off invading viruses and bacteria. However, recent work suggests that our immune systems may also being fighting a war against fat.

When laboratory mice are engineered to lack a specific type of immune cell, they become obese and show signs of high blood pressure, high cholesterol, and diabetes. Even though these findings have yet to be replicated in humans, they are already helping scientists understand the triggers of metabolic syndrome, a cluster of conditions associated with obesity.

A new study “definitely moves the field forward,” says immunologist Vishwa Deep Dixit of the Yale School of Medicine, who was not involved in the work. “The data seem really solid.”

Scientists have known for some time now that there is a correlation between inflammation—a heightened immune response—and obesity. Fat cells have the ability to release inflammatory molecules, which complicates these findings, since it is difficult to distinguish if the inflammation causes weight gain or is a side effect of weight gain.

Immunologist Yair Reisner of the Weizmann Institute of Science in Rehovot, Israel, came upon this new cellular link between obesity and the immune system while he was studying autoimmune diseases. Reinser was interested in an immune molecule called perforin, which kills diseased cells by boring a hole in their outer membrane. Reisner’s group suspected that perforin-containing dendritic cells might also be destroying the body’s own cells in some autoimmune diseases. To test their hypothesis, Reisner and his colleagues engineered mice that lacked perforin-wielding dendritic cells. Then they waited to see whether they developed any autoimmune conditions.

“We were looking for conventional autoimmune diseases,” Reisner says. “Quite surprisingly, we found that the mice gained weight and developed metabolic syndrome.”

Mice lacking the dendritic cells with perforin had high levels of cholesterol, early signs of insulin resistance, and molecular markers in their bloodstreams associated with heart disease and high blood pressure. Furthermore, the immune systems of these laboratory animals revealed that they also had a peculiar balance of T cells—a type of white blood cell that directs immune responses.

Reisner and his colleagues report online in the journal Immunity that when they removed these T cells from the mice, the absence of dendritic cells no longer caused the animals to become obese or develop metabolic syndrome.

The results, according to Reisner, suggest that the normal role of the perforin-positive dendritic cells is to keep certain populations of T cells under control. In the same way that perforin acts to kill cells infected with viruses, it can be directed to kill subsets of unnecessary T cells. When the brakes are taken off those T cells, they cause inflammation in fat cells, which leads to altered metabolism and weight gain.

“We are now working in human cells to see if there is something similar going on there,” Reisner says. “I think this is the beginning of a new focus on a new regulatory cell.” If these results turn out to be true in humans, they could point toward a way to use the immune system to treat obesity and metabolic disease.

Daniel Winer, an endocrine pathologist at the University of Toronto in Canada and the lead author of a January Diabetes paper that links perforin to insulin resistance, says the new results overlap with his study. Winer and his group found that mice whose entire immune systems lack perforin developed the early stages of diabetes when fed a high-fat diet. This new paper builds on that by homing in on perforin-positive dendritic cells and showing the link even in the absence of a high-fat diet. “It provides further evidence that the immune system has an important role in the regulation of both obesity and insulin resistance.”

Even if the results hold true in humans, however, a treatment for Type 2 diabetes, obesity or metabolic disease are far off. Dixit said. “Talking about therapeutics at this point would be a bit of a stretch.” Injecting perforin into the body could kill cells beyond those T cells that promoting obesity. We can’t live without any T cells at all, since they are vital to fight diseases and infections.

However, research on what these T cells are recognizing when they seek out fat cells and cause inflammation in fat tissue could eventually reveal drug targets.

Using Cells from the Mouth to Cure Blindness


If we take tissue samples from the mouth and grow them in the laboratory and manipulate them, we might be able to cure the blind. Blind people who suffer from stem cell deficiency in the cornea might be able to see again by using stem cells isolated from the mouth. Furthermore, this treatment might not only restore vision, but it might also ameliorate pain in the cornea.

Ophthalmologist Tor Paaske Utheim has conducted research for over ten years on how to cure certain types of blindness by using stem cells harvested from tissue obtained from different parts of the body. He then transplants this cultured tissue into the damaged eye, and patients who suffer from blindness as a result of corneal stem cell deficiencies can regain their sight. Recently, Utheim’s research has utilized stem cells from the mouth to grow new corneal tissue, and has also tried to design optimal methods to store and transport this tissue to treat patients.

Utheim is the head of a research group at the Faculty of Dentistry at the University of Oslo (UiO) and the Department of Medical Biochemistry at Oslo University Hospital.

Using cells extracted from the mucous membrane lining the inside of the mouth (the oral mucosa) can restore vision is new to most people. Only ten years ago, this was considered impossible, but results confirm the potential of this method. Twenty clinical studies from various countries have, to date, shown good results, according to Utheim. These clinical trials, however, have only applied these cells to a group of diseases caused by stem cell deficiency in the cornea.

Utheim and his colleagues hope to treat patients with eye injuries caused by so-called limbal stem cell deficiencies. This disorder can be caused by such things as UV radiation, chemical burns, serious infections like trachoma, and various other diseases, some of which are heritable. The number of people worldwide affected by limbal stem cell deficiency is unknown, but in India alone there is an estimated 1.5 million. This disorder most often affects people living in developing countries.

Stem cells that are found at the outer edge of the cornea help to keep the surface of the cornea even and clear. In limbal stem cell deficiencies, the stem cells have been damaged, and they cannot renew the cornea’s outermost layer. Instead, other cells grow over the cornea, which clouds the cornea. The cornea can become fully or partially covered, explains Utheim, which leads to impaired vision or blindness.

The stem cells are localized in the periphery of the cornea; an area known as the limbus.  They=se limbal stem cells renew the outermost layer of the cornea. Illustration: Amer Sehic, OD/UiO.
The stem cells are localized in the periphery of the cornea; an area known as the limbus. These limbal stem cells renew the outermost layer of the cornea. Illustration: Amer Sehic, OD/UiO.

Others suffer from severe pain as well. When one patient was interviewed by Norwegian national broadcaster NRK about his limbal stem cell deficiency, he responded: “I don’t know what’s worse: the pain, or losing my sight.”

Utheim explained that when stem cells do not work properly, ulcers can develop in the cornea, which exposed nerve fibers. Since the number of nerve fibers is far higher in the cornea than for example in the skin, it is not surprising that some patients experience severe pain.

Eyes that suffer mildly from limbal stem cell deficiency. The stem cells stall and other cells grow over the cornea. The window of the eye, normally clear and transparent, is thus blurred, leading to reduced vision.Photo: Dr. Takahiro Nakamura, Department of Ophthalmology/Kyoto Prefectural University of Medicine.
Eyes that suffer mildly from limbal stem cell deficiency. The stem cells stall and other cells grow over the cornea. The window of the eye, normally clear and transparent, is thus blurred, leading to reduced vision. Photo: Dr. Takahiro Nakamura, Department of Ophthalmology/Kyoto Prefectural University of Medicine.

A breakthrough within the field occurred about ten years ago when Japanese researchers showed that cells from the oral mucosa could be used to replace limbal stem cells in patients with limbal stem cell deficiency. Although it had been possible since the late 1990s to cure the disorder using cultured stem cells. The available treatment relied on the patient having a healthy eye from which to collect cells.

Further developments made it possible to harvest cells from a relative or deceased individual, but using limbal stem cells from other patients required the use of strong immunosuppressive drugs for the patients, which could cause serious side effects.

A milestone seemed to be reached when it became possible to use a patient’s own cells to treat blindness in both eyes without the need for immunosuppressive drugs. Strangely, this makes some sense because there are similarities between the oral mucosae and the surface of the eye (see Utheim TP. Stem Cells. 2015;33:1685-1695). Originally, using mouth mucosal cells to treat the eye required that the laboratory where the cells are cultured and the clinic where the patients are treated be quite close together. Because there were no protocols for storing extracted oral mucosal cells so that they can be easily kept and transported. This has made the treatment virtually inaccessible to many of the patients who need it the most, namely those in developing countries. However, this may be about to change.

Utheim’s research group is now on the brink of a development that will make it possible to cure both severe pain and blindness in patients who are spread over a larger geographical area than before (see Islam R, et al. PLoS One. 2015;10:e0128306.). “Today, cells from the mouth are cultured for use in the treatment of blindness in only a few specialized centers in the world. By identifying the optimal conditions for storing and transporting the cultured tissue, we would allow for the treatment to be made available worldwide, and not just close to the cell culture centers,” said Rakibul Islam, who is a PhD candidate in the Department of Oral Biology at the Faculty of Dentistry.

Islam is collaborating with Harvard Medical School to introduce this method of treating blindness to clinics around the world. Islam’s findings could also help improve treatment outcomes. “Being able to store the cultured tissue in a small sealed container for a week increases the technique’s flexibility significantly. It makes it easier to plan the operation and allows for quality assurance through microbiological testing of the tissue before transplantation,” Islam explained.

One of the things that Islam and his colleagues have discovered is the specific temperature range at which cells from the mouth should ideally be stored at after culture. Islam has shown that cultured mouth stem cells retain their quintessential properties best between 12 and 16 degrees Celsius (See Dolgin, Elie. Nature Biotechnolgy, 2015;33:224-225.).

During a brief stint at Harvard University, Islam also examined which areas of the mouth are best suited to use in regenerative medicine. In other words, Islam and his colleagues wanted to know which parts of the mouth contain cell layers that regenerate the fastest. Islam explained this using this example: “If you burn any part of your mouth on hot coffee, it heals so quickly that by the next morning you have forgotten about it. This is because the oral mucosa contains cells that multiply quickly. We wanted to investigate whether there were regional differences in the mouth that we could exploit for the treatment of limbal stem cell deficiency.”

Islam continued, “Our results show that the location from which the mucosal tissue is harvested has a striking impact on the quality of the cultured tissue.”

The results from this particular study have not yet been published.

This research can potentially give hope to the many blind that live far away from centralized cell culture laboratories.  In work by Utheim in 2010, in collaboration with the ophthalmologist Sten Ræder, he developed storage technology for cultured stem cells that enables the cultured tissue to be transported in a small custom-made plastic container.  Tissue from stem cells is thus freed from expensive and bulky laboratory equipment and provides a whole new level of flexibility.

Utheim said “The sample of cells from the mouth can be sent by air over long distances to specialist laboratories with first-class equipment and expertise. After a couple of weeks of laboratory cultivation, the sender may receive the tissue back ready for use. An ophthalmologist could then transplant the stem cells onto the patient’s eye.”

However, the container was just one step in the right direction: “Now we have identified those areas of the mouth that may be best suited for regenerative medicine, and developed a method for storing and transporting tissue from centralized, highly specialized tissue culture centers to clinics worldwide. Our findings are helping to simplify and streamline the clinical procedures, and to make the treatment far more accessible than it is today,” said Islam, who admitted that the transport potential of the project has been integral to his own enthusiasm.  He continued, “Although the scientific and technical aspects of our project are very exciting, it has been especially motivating to think of the possibilities this storage technology brings to treating blindness in all parts of the world, including my homeland Bangladesh.”

A central laboratory for the growth of stem cells already exists in Italy.  In fact, earlier this year the European Medicines Agency approved the procedure for the cultivation of stem cells from the cornea in EU laboratories. This is the first stem cell therapy to be approved by the European Medicines Agency, according to the journal Nature Biotechnology.  Utheim described the approval as an important step towards the implementation of stem cell technology over larger geographical areas.  To date, almost 250 people with limbal stem cell deficiency have undergone treatment involving transplantation of stem cells grown from their own mouth cells.  “This provides a good basis for judging the success of the treatment” Utheim says.

He has recently published an article in the journal Stem Cells on the inherent potential of cells from the mouth to regenerative medicine.  Roughly three out of four treatments are described as successful.