Protein Regulates Heart Muscle Development

Scientists from the Center for Genomic. Regulation in Barcelona, Spain have discovered a genetic regulatory network that revolves around a protein called Mel18. This regulatory network acts as a genetic switch during the differentiation of embryonic stem cells into heart muscle cells.

Mel18 acts in combination with a vitally important set of proteins called the “Polycomb Regulatory Complexes” or PRCs. PRCs are probably one of the major repressors of genes in adult and embryonic stem cells, and in this paper, Luciano De Croce and his colleagues showed that Mel18 acts with the PRCs to suppress gene expression.

Beyond that, however, once differentiation occurs, Mel18 combines with other proteins to continue to shut off the expression of unnecessary genes, but during early cardiac development, Mel18 completely shifts and becomes a driver of gene expression. It shifts its function by forming new complexes with other proteins that regulate gene expression in various ways.

Graphical abstract_5x5

Thus Mel18 acts as a genetic switch that guides stem cells into the cardiac fate and eventually into the heart muscle cell lineage.

This fascinating work, which was published in the journal Cell Stem Cell, can help stem cell scientists grow better heart muscle from induced pluripotent stem cells in the laboratory. It could also elucidate the underlying causes of heart defects in congenital heart disease. They may also lead to new ways of controlling stem cells in the laboratory to grow cellular repair kits and patches for patients with damaged or sick hearts.

New Gene Therapy for Retinitis Pigmentosa Treats Early and Late Stages of the Disease in Dogs

Collaboration between scientists from the University of Pennsylvania and the University of Florida, Gainesville has hit pay dirt when it comes to treating an inherited eye disease. This study used gene therapy to treat the disease and the results of this research project make a definitive contribution to the development of gene therapies for people with the blinding eye disorders for which there is presently no cure.

The disease in question is called retinitis pigmentosa, which is a group of rare, genetic disorders characterized by the degradation and subsequent loss of photoreceptors in the retina. People who suffer from retinitis pigmentosa have difficulty seeing at night and experience a loss of peripheral vision.

As mentioned, retinitis pigmentosa is an inherited disorder that results from mutations in any one of more than 50 different genes. These genes encode proteins that are required for retinal photoreceptors, and mutations in these genes compromises photoreceptor survival and function.

In human patients, retinitis pigmentosa is the most common inherited disease that results in degeneration of the photoreceptors of the retina. Approximately 1 in 4,000 people are affected with retinitis pigmentosa and 10 to 20 percent have a particularly severe form called X-linked retinitis pigmentosa. This disease predominately affects males, who experience night blindness by age 10 and progressive loss of the visual field by age 45. 70 percent of people with the X-linked retinitis pigmentosa harbor loss-of-function mutations in the retinitis pigmentosa GTPase Regulator (RPGR) gene. RPGR encodes a protein that maintains the health and survival of retinal photoreceptors. There are two types of photoreceptors; rods that give us the ability to see in dim light, and cones that allow us to see fine detail and color in bright light. Loss of the RPGR protein damages both types of photoreceptors.

Because there are no treatments for retinitis pigmentosa, gene therapy might be the best option to treat this disease. Fortunately, some varieties of dogs have a naturally occurring, late-stage retinitis pigmentosa that closely resembles the human disease. In previous experiments, gene therapies were used in diseased dogs, but such studies showed that benefits from gene therapy were only observed when it was used in the earliest stages of the disease.

“The study shows that a corrective gene can stop the loss of photoreceptors in the retina, and provides good proof of concept for gene therapy at the intermediate stage of the disease, thus widening the therapeutic window,” said Neeraj Agarwal, Ph.D., a program director at National Eye Institute, a part of the National Institutes of Health, who funded this research.

The dogs used in this study all suffered from a naturally occurring canine form of RPGR X-linked retinitis pigmentosa that is observed in some mixed breeds. These animals provided an excellent model system for their gene therapy tests, since affected dogs with early to late stages of the disease could be treated with the experimental therapy in one eye while the other untreated eye could be evaluated in parallel as a control.

To treat these blind dogs, the team utilized adeno-associated virus (AAV). They engineered AAV particles that possessed the entire RPGR gene. Then they devised a way to deliver these viruses to the retinal cells so that the viruses could infect the retinal cells and produce normal copies of the RPGR protein.

When the eyes treated with the AAV vectors were subjected to detailed imaging, it was clear that the gene therapy protocol arrested the thinning of the retinal layer. This shows that the treatment halted the degeneration of the photoreceptors in the affected dogs. When the treated eyes were compared with the untreated eye, the structure of the rod and cone photoreceptors was obviously improved and better preserved in the treated eye in comparison to the untreated eye. When the neural physiology of the retinas from the treated and untreated eyes was compared, once again, the retinas from the eyes treated with the gene therapy were more normal than the untreated eyes. In fact, the gene therapy halted the photoreceptor cell death associated with retinitis pigmentosa for two and a half years, which was the length of the study.

The team also treated dogs who suffered from later-stage disease in the hope that the gene therapy could not only improve the condition of dogs in the early stages of the disease, but also those with later stages of the disease. Interestingly, the gene therapy also froze the loss of retinal thickness and preserved the structure of surviving photoreceptors, but the retinas in the untreated eyes continued to thin and their photoreceptor function deteriorated as well. When the dogs were sent through an obstacle course and a maze under dim light, the animals did significantly better when they used their eye that had been treated with the gene therapy compared with their performance when they used the untreated eye. This shows that this gene therapy also works in dogs suffering from the late-stages of retinitis pigmentosa.

Can such a therapy be used in people in human clinical trials? Not yet. More safety testing must be done in order to properly determine if it is safe over long periods of time, according to this study’s co-leaders, Gustavo Aguirre, V.M.D., Ph.D., and William Beltran, D.V.M., Ph.D., of the University of Pennsylvania. Other collaborators, University of Pennsylvania scientists Artur Cideciyan, Ph.D., and Samuel Jacobson, M.D., Ph.D. are presently screening potential patients who have RPGR mutations as a prolegomena for a future clinical trial.

Their results are published in Proceedings of the National Academy of Sciences.

Pre-treatment of MSCs Can Reduce Their Regenerative Properties

Mesenchymal stem cells (MSCs) are excellent suppressors of unwanted inflammation.  This anti-inflammatory activity has been established for systemic inflammatory diseases in animal experiments (Klinker MW, Wei CH. World J Stem Cells. 2015 Apr 26;7(3):556-67), and in clinical trials with human patients (Dulamea A. J Med Life. 2015 Jan-Mar;8(1):24-7; Simonson OE et al., Stem Cells Transl Med. 2015 Oct;4(10):1199-213. doi: 10.5966/sctm.2015-0021).  Stem cell researchers have also shown that MSCs can suppress inflammation in the bowel (see Swenson E and Theise N. Clinical and Experimental Gastroenterology 2010;3:1-10; Chen Z, et al., Biochem Biophys Res Commun. 2014 Aug 8;450(4):1402-8).

After being introduced into the body of a patient, MSCs to move to the site where they are needed (a phenomenon known as “homing”) and promote tissue repair and healing.  Sometime MSC homing works quite well, but other times, it is so-so.  Therefore, several inventive scientists have devised ways to beef up homing to specific sites in order to improve MSC-based tissue healing.  Also, investigators are equally interested in increasing the ability of MSCs to stick to tissues once they arrive there to ensure that the homed MSCs stay where they are needed (see Kavanagh DP, Robinson J, and Kalia N. Stem Cell Rev 2014;10:587-599).  Unfortunately, at the moment, the whole homing process is a bit of a black box and while artificially increasing homing might help in the laboratory, whether or not it increases the therapeutic benefit of MSCs is even less well understood.

A new report from the laboratory of Neena Kalia, who works at the University of Birmingham, UK, has examined the effect of artificial enhancement on the therapeutic capacity of MSCs to treat inflammation in the bowel.  This is an important study because pre-treatment strategies have been suggested as ways to boost MSC homing and retention to various tissues.  The Kalia study suggests such pre-treatment strategies should be viewed with a degree of skepticism.

In this study, Kalia her coworkers induced inflammation in the gastrointestinal tracts of mice by clamping off the blood supply to the this tissue for a time and then releasing the clamps and letting the blood flow anew.  This type of damage, known as ischemia/reperfusion (IR) injury deprives cells of vital oxygen and nutrients for a short period of time, which causes some cells to die.  When the blood is allowed to flow into the tissue, inflammation is induced in the damaged tissue.  Therefore, this technique can efficiently induce  inflammation in tissues in the gastrointestinal tract.

Two groups of mice were treated with bone marrow-derived MSCs.  One group had experiences IR injury to their gastrointestinal tracts, and the other group did not.  In these experiments, administered MSCs showed similar levels of and cell adhesion in both injured and non-injured guts.  In general, cell adhesion levels were nothing to write home about:  as reported in the paper, “limited cell adhesion observed.”  Despite these initial observations, those MSCs that found their way to the gut were able to help heal the tissues to some degree.  There were fewer white blood cells in the middle part of the small intestine (jejunum), and the degree of blood flow seemed to have improved.  Unfortunately, the lower part of the small intestine (ileum) was not helped to the same degree, and the paper reports that a fair number of MSCs got stuck in small blood vessels, which suggests that these vessels got stuck on their way to the intestine.

If these results seem underwhelming, it might be because they are.  Undaunted, Kalia and her crew tried to boost the regenerative abilities of their isolated MSCs by pretreating them.  Kalia’s laboratory and other laboratories as well have used a variety of chemical agents to augment the healing abilities of MSCs.  These agents include things like tumor necrosis factor (TNF)-α, CXCL12 (also known as stromal cell-derived factor 1 or SDF1, which strongly activates white blood cells), interferon (IFN)-γ, or hydrogen peroxide.  When these pre-treated MSCs were administered to mice whose guts were damaged by means of IR injury, the pretreatment not only did not enhance their intestinal recruitment, but actually decreased the healing capacities of MSCs.  Pretreatment of MSCs with tumor necrosis factor (TNF)-α, CXCL12, interferon (IFN)-γ, or hydrogen peroxide did not enhance their intestinal recruitment.  Pretreatment with TNFα and IFNγ abrogated ability of transplanted MSCs to reduce white blood cells infiltration and improve blood flow in the jejunum.

Kalia and her colleagues utilized a technique called “intravital” microscopy for this study.  Intravital microscopy can track individual cells in a living animals (Kavanagh DP, Yemm AI, Zhao Y, et al. PLoS One 2013;8:e59150). With this technique, they were able to efficiently monitor adhesion in the tinyu blood vessels in the injured intestinal tissue.  They documented poor MSC adhesion to the gut lining and that pre-treatment with various factors hopes failed to enhance adhesion of MSCs to the gut.

This study successfully demonstrated that MSCs can rapidly limit white blood cells recruitment to the inflamed gut, and improve tissue perfusion if they are administered after intestinal IR injury. However, Kalia’s study also shows that strategies to improve MSC therapeutic efficacy by means of pretreatment of MSCs may not be all it’s cracked up to be.  They suggest that in the future, cytokine or chemical pretreatments designed to enhance MSC recruitment and function will require more than just successful experiments in a cell culture system.  Instead, pretreatment strategies will need to be carefully validated in living organisms in order the confirm that such protocols help rather than hinder the therapeutic function of implanted stem cells.

This paper was published in the journal Stem Cells – Kavanagh DP, Suresh S, Newsome PN, et al. Stem Cells 2015;33:2785-2797

A New Target for Treating Stroke: The Spleen

If the blood vessels of the brain become plugged as a result of a clot or some other obstructive event, then the brain suffers a trans-ischemic attack (TIA), which is more commonly known as a stroke. The initial stroke starves brain cells of oxygen, which causes cell death by suffocation. However, dying brain cells  often spill enormous amounts of lethal material into the surrounding area, which kills off even more brain cells. Worse still, these dead or dying called can induce inflammation in the brain, which continues to kill off brain cells.

New work, however, from the laboratory of César Borlongan at the University of Southern Florida in Tampa, indicates that the spleen may be a target for treating the stroke-induced chronic inflammation that continues to kill brain cells after the initial stroke.

At the University of Florida Center of Excellence for Aging and Brain Repair, a study found that the intravenous administration of human bone marrow stem cells to post-stroke rats reduced the inflammatory-plagued secondary cell death associated with stroke progression in the brain. The intravenously administered cells preferentially migrated to the spleen where they reduced this post-stroke inflammation.

This study answers some of the perplexing questions surrounding animal experiments that used stem cells to treat stroke. Typically, stem cell administration to animals that suffered an artificially-induced stroke causes some functional recovery, but when their brains are examined for the stem cells that were implanted into them, very few surviving cells are observed.

“Our findings suggest that even if stem cells do not enter the brain or survive there, as long as the transplanted cells survive in the spleen the anti-inflammatory effect they promote may be sufficient enough to therapeutically benefit the stroke brain,” said César Borlongan, principal investigator of this study.

Stroke is the leading cause of death and the number one cause of chronic disability in the United States, yet treatment options are limited.

Stem cell therapy has emerged as a potential treatment for ischemic stroke, but most pre-clinical studies have examined the effects of stem cells transplanted during acute stroke (one hour to three hours aster the onset of the stroke).

In the wake of an acute stroke, an initial brain lesion forms from the lack of blood flow to the brain. The blood-brain barrier is also breached and this allows the infiltration of inflammatory molecules that trigger secondary brain cell death in the weeks and months that follow. This expanded inflammation is the hallmark of chronic stroke.

In this study, Borlongan and his colleagues intravenously administered human bone marrow stem cells 60 days after the onset of a stroke. Thus these animals were well into the chronic stroke stage.

The transplanted stem cells predominantly homes to the spleen. In fact, Borlongan and his crew found 30-times more cells in the spleens of the animals than in the brain.

While in the spleen, the stem cells squelched the production of a protein called tumor necrosis factor, which is a major inflammatory signal that increases in concentration after a stroke. The reduction of the tumor necrosis factor signal prevented the macrophages and other immune cells from leaving the spleen and going to the brain. This reduced systemic inflammation and decreased the size of the lesions in the brain caused by the stroke. There was also a trend toward reduced neuronal death and smaller decreases in learning and memory in the laboratory animals.

Borlongan explained that during the chronic stage of stroke, macrophages seem to fuel inflammation. “If we can find a way to effectively block the fuel with stem cells, then we may prevent the spread of damage in the brain and ameliorate the disabling symptoms many stroke patients live with,” said Borlongan.

Borlongan and his team hope to test whether transplanting human bone marrow stem cells directly into the spleen will lead to behavioral recovery in post-stroke rats.

One drug that has been approved for the emergency treatment of stroke is tPA or tissue plasminogen activating factor, which activates the blood-based protein plasminogen to form the highly active enzyme, plasmin. Plasmin is a powerful dissolved of clots, but tPA must be administered less than 4.5 hours after the onset of ischemic stroke, and benefits only three to four percent of patients.

Even though more work needs to be done, evidence from the USF group and other neurobiology groups indicates that stem fells may provide a more effective treatment for stroke over a wider time frame.

Targeting the spleen with stem cells or the anti-inflammatory molecules they sec rate offers hope for treating chronic neurodegenerative diseases like stroke at later stages.

This study, which was published in the journal Stroke, shows that it is possible to arrest the chronic inflammation that characterizes chronic stroke 60 days after the initial stroke. If such a result can be replicated in human patients, it will indeed be a powerful thing, according the Sandra Acosta, the first author on this paper.

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.

Rejection of Induced Pluripotent Stem Cell Derivatives By the Immune System is a Function of Where They are Transplanted

Induced pluripotent stem cells (iPSCs) are made from mature, adult cells by a combination of genetic engineering and cell culture techniques. Master genes are transfected into mature cells, which are then cultured as they grow and revert to more immature states. Eventually, a population of cells grow in culture that have some, though not all of the characteristics, of embryonic stem cells. Because these cells are pluripotent, they should, theoretically have the ability to differentiate into any adult cell type. Also, since they are derived from a patient’s own cells, they should be tolerated by the patient’s immune system and should not experience tissue rejection.I

Or should they? Experiments with cells derived from iPSCs have generated mixed results. If C57BL/6 (B6) mice are transplanted with iPSC-derived cells, such cells show some levels of recognition by the immune system. However, another study has concluded that various lineages of B6 iPSC-derived cells are not recognized by the immune system when transplanted under the kidney capsule of B6 mice. Why the contradiction?

Yang Xu and his colleagues at the University of California, San Diego have attempted to resolve this controversy by utilizing a mouse model system. Xu and his colleagues used the same B6 transplantation model and transplanted a variety of different cells derived from iPSCs that were made from cells that came from the same laboratory mice.

Xu and others showed that iPSC-derived and embryonic stem cell (ESC)-derived cells are either tolerated or rejected, depending upon WHERE they are transplanted. You see the immune system depends upon a network of cells called “dendritic cells” to sample the fluids that circulate throughout the body and identify foreign substances. Some locations in our bodies are chock-full of dendritic cells, while other locations have a paucity of dendritic cells. When iPSC or ESC-derived cells are transplanted under the kidney capsule, they survive and thrive. The kidney capsule has a distinct lack of dendritic cells. However, if these same cells, which were so nicely tolerated under the kidney capsule, are transplanted under the skin or injected into muscles, they were rejected by the immune system. Why? These two sites are loaded with dendritic cells.

Therefore, the rejection of iPSC-derived cells by the patient’s body is more of a function of where the cells are transplanted than the cells themselves. Mind you, poor quality iPSCs can produce derivatives that are rejected by the immune system, but high-quality iPSCs can differentiate into cells that are accepted by the immune system, but it is wholly dependent on where they are transplanted.

Perhaps, transplanted IPSC derivatives will need the immune system suppressed for a short period of time and after they become integrated into the patient’s body, the immune suppression can be lifted. Alternatively it might be possible to induce tolerance to the transplanted cells with immunological tricks. Either way, understanding why iPSCs-derived cells are rejected or accepted by the patient’s immune system is the next step to using these amazing cells for regenerative medicine.

Xu’s paper appeared in the journal Stem Cells – DOI: 10.1002/stem.2227.