Using Drugs to Stimulate your Own Stem Cells to Treat Multiple Sclerosis


Paul Tesar from Case Western Reserve in Cleveland. Ohio and his colleagues have discovered that two different drugs, miconazole and clobetasol, can reverse the symptoms of multiple sclerosis in laboratory animals. Furthermore, these drugs do so by stimulating the animals’ own native stem cell population that insulates nerves.

Multiple sclerosis (MS) is a member of the “demyelinating disorders.” The cause of MS remains unknown, but all of our available evidence strongly suggests that MS is an autoimmune disease in which the body’s immune system attacks its own tissues. In MS the immune system attacks and destroys myelin — the fatty substance that coats and protects nerve fibers in the brain and spinal cord. We can compare myelin to the insulation that surrounds electrical wires. When myelin is damaged, the nerve impulses that travel along that nerve may be slowed or blocked.

The myelin sheath is made by cells known as “oligodendrocytes,” and oligodendrocytes are derived from a stem cell population known as OPCs, which stands for oligodendrocyte progenitor cells. If this stem cell population could be stimulated, then perhaps the damaged myelin sheath could be repaired and the symptoms of MS ameliorated.

In a paper that appeared in the journal Nature (522, 2015 216-220), Tesar and the members of his research team, and his collaborators used a pluripotent mouse stem cell line and differentiated them into OPCs. Thyroid hormone is a known inducer of OPC differentiation. Therefore, Tesar and others screened a battery of drugs to determine if any of these compounds could induce OPC differentiation as cell as thyroid hormone. From this screen using cultured OPCs, two drugs, the antifungal drug miconazole and clobetasol, a corticosteroid of the glucocorticoid class, proved to do a better job of inducing OPC differentiation than thyroid hormone.

Was this an experimental artifact? Tesar and others devised an ingenious assay to measure the effectiveness of these two drugs. They used brain slices from fetal mice that were taken from animals whose brains had yet to synthesize myelin and applied OPCs to these slices with and without the drugs. With OPCs, no myelin was made because the OPCs did not receive any signal to differentiate into mature oligodendrocytes and synthesize myelin. However in the presence of either miconazole or clobetasol, the OPCs differentiated and successfully myelinated the brain slices.

Experiments in tissue culture are a great start, but do they demonstrate a biological reality within a live animal? To answer this question, Tesar and his crew injected laboratory mice with purified myelin. The immune systems of these mice generated a robust immune response against myelin that eroded the myelin sheath from their nerves. This condition mimics human MS and is called experimental autoimmune encephalitis, and it is an excellent model system for studying MS. When mice with experimental autoimmune encephalitis (EAE) were treated with either miconazole or clobetasol, the EAE mice showed a remarkable reversal of symptoms and a solid attenuation of demyelination. Tissue samples established that these reversals were due to increased OPC activity.

When the mechanisms of these drugs were examined in detail, it became clear that the two drugs worked through distinct biochemical mechanisms. Miconazole, for example, activated the mitogen-activated protein kinase (MAPK) pathway, but clobetasol worked through the glucocorticoid receptor signaling pathway. Both of these signaling pathways converge, however, to increase OPC differentiation.

Both miconazole and clobetasol are only approved for topical administration. However, the fact that these drugs can cross the blood-brain barrier and effect changes in the brain is very exciting. Furthermore, this work establishes the template for screening new compounds that might be efficacious in human patients.

In the meantime, human patients might benefit from a clinical trial that determines if the symptoms and neural damage caused by MS can be reversed by the administration of these drugs or derivatives of these drugs.

Temple University Lab Shows Exosomes from Stem Cells Heal Hearts After a Heart Attack


Temple University stem cell researcher Raj Kishore, who serves as the Director of the Stem Cell Therapy Program at the Center for Translational Medicine at Temple University School of Medicine (TUSM), and his colleagues have used exosomes from stem cells to induce tissue repair in the damaged heart. The results of this fascinating research made the cover of the June 19, 2015 edition of the leading cardiovascular research journal, Circulation Research.

“If your goal is to protect the heart, this is a pretty important finding,” Dr. Kishore said. “You can robustly the heart’s ability to repair itself without using the stem cells themselves. Our work shows a unique way to regenerate the heart using secreted vesicles from embryonic stem cells.” Kishore’s group is in the early stages of characterizing the molecules in these exosomes that are responsible for inducing and potentiating tissue repair.

The heart beats throughout the lifetime of an individual. Despite its apparent constancy, the heart possess little to no ability to repair itself. When heart muscle is damaged in a heart attack, the heart is unable to replace the dead tissue and grow new contracting heart muscle. Instead, after a heart attack, it compensates for lost pumping ability by enlarging, a phenomenon known as ”remodeling.” .Remodeling, however, come with a high price, since the heart grows beyond the ability of the sparse cardiac circulatory system to properly convey blood to the enlarged heart muscle. Consequently, heart contraction weakens, leading to a condition known as congestive heart failure, which contributes to, or causes one in nine deaths in the United States. Heart disease is our nation’s leading killer.

Given the fact that heart disease is the result of the death of heart muscle cells, this condition seems to tailor-made for stem cell therapy. A variety of animal experiments with stem cells from bone marrow, muscle, fat, or embryos have shown that stem cells can regenerate heart muscle. However, the regeneration of the heart is much more complicated than was originally thought. For example, injecting damaged hearts with stem cells turned out to be a rather ineffective strategy because the heart, after a heart attack, is a very hostile place for newly infused cells. Dr. Kishore noted, “People know if they inject hundreds of stem cells into an organ, you’re going to be very lucky to find two of them the next day. They die. It’s as though you’re putting them into the fire and the fire burns them.”

Dr. Kishore has used a very different approach for regenerative medicine. Over 10 years ago, cancer researchers discovered tiny sacks excreted by cells that they called “exosomes.” These exosomes were thought to be involved with waste disposal, but later work showed that they were more mini-messengers, carry telegrams between cells. Exosomes proved to be one way a primary tumor communicated with distant metastases. Researchers later discovered that nearly all cell types excrete exosomes. Dr. Kishore and his team began to study the exosomes of stem cells to determine if these small vesicles could solve the heart-repair problem.

In 2011, Kishore’s team published the first paper to ever examine stem cell exosomes and heart repair. This paper established Kishore and his research team as a pioneer in exosome research and in the use of exosomes in the treatment of heart disease. A year after that paper, there were a total of 52 papers published on exosomes, but today there are 7,519 papers reporting on exosome research. Among those studies, only 13 or 14 have examined exosomes in heart disease. This new paper by Dr. Kishore’s team marks its third contribution to the science of exosomes and heart repair.

In the current study, Kishore and others used a mouse model of heart attack. Also involved in the research are Dr. Kishore’s colleagues from Temple’s Center for Translational Medicine, the Cardiovascular Research Center, and the Department of Pharmacology, as well as researchers from the Feinberg Cardiovascular Research Institute at Northwestern University in Chicago.

In this study, after suffering a heart attack, the mice received exosomes from either embryonic stem cells or exosomes from fibroblasts. Mice that received the fibroblast-derived exosomes served as the control group. The results were unmistakable. Mice that received exosomes from embryonic stem cells showed significantly improved heart function after a heart attack compared to the control group. More heart muscle cells in these mice survived after the heart attack, and their hearts also exhibited less scar tissue. Fewer heart cells committed suicide — a process known as programmed cell death, or apoptosis. Also, hearts from mice treated with embryonic stem cell-derived exosomes showed greater capillary development around the areas of injury. The increased density of blood vessels improved circulation and oxygen supply to the heart muscle. Further, there was a marked increase in endogenous cardiac progenitor cells, which is the hearts own internal stem cell population. These cells survived and created new heart cells. The heartbeat was more powerful in the experimental group compared to the control group, and the kind of unhealthy enlargement that compensates for tissue damage was minimized.

Vishore’s group also tested the effect of one of the most abundant gene-regulating molecules (microRNAs) found in the stem cell exosome; a microRNA called miR-294. When purified miR-294 alone was introduced to cardiac stem cells in the laboratory, it mimicked many of the effects seen when the entire exosome was delivered. “To a large extent, this micro-RNA alone can recapitulate the activity of the exosome,” Dr. Kishore said. “But we can never say it is responsible for all of the response because embryonic stem cell exosomes have many other microRNAs.”

Future research will examine both exosome therapy and the use of specific microRNAs for heart repair in large-animal models of heart attack with a view to eventually testing these components in human patients in clinical trials.

“Our work shows that the best way to regenerate the heart is to augment the self-repair capabilities and increase the heart’s own capacity to heal,” Dr. Kishore said. “This way, we’re avoiding risks associated with teratoma formation and other potential complications of using full stem cells. It’s an exciting development in the field of heart disease.”

Gene Therapy Increases Stem Cell Recruitment to Heart and Improves Heart Function


Data from a Phase 2 clinical trial is creating quite a stir in cardiology circles. According to the findings of this study, the single administration of a gene on a non-viral-derived plasmid improves cardiac structure, function, serum biomarkers and clinical status in patients with severe ischemic heart failure one year after treatment.

The results from the final 12-months of the Phase 2 STOP-HF clinical trial for the JVS-100 treatment were presented at the European Society of Cardiology – Heart Failure 2015 meeting by the developer of this technology: Juventas Therapeutics Inc. The founder of Juventas, Marc Penn, M.D., Ph.D., FACC, is also the medical officer and director of Cardiovascular Research and Cardiovascular Medicine Fellowship at Summa Health in Akron, Ohio. Dr. Penn presented the results of this randomized, double-blind, placebo-controlled STOP-HF trial, which included treatments on 93 patients at 16 different clinical centers in the United States.

“The results from STOP-HF demonstrate that a single administration of 30 mg of JVS-100 has the potential to improve cardiac function, structure, serum biomarkers and clinical status in a population with advanced chronic heart failure who are symptomatic and present with poor cardiac function,” stated Dr. Penn. “These findings combined with our deep understanding of SDF-1 biology will guide future clinical trials in which we plan to prospectively study the patient population that demonstrated the most pronounced response to JVS-100. In addition, we will further our understanding of JVS-100 by determining if a second administration of drug may enhance benefits beyond those we observed with a single administration.”

These study. some patients received a 30 mg dose of JVS-100 while others received a placebo.  Patients who received JVS-100 showed definite improvements 12 months after treatment.  The cardiac function and heart structure of the patients who received JVS-100 were far better than those who had received the placebo.  JVS-100-treated patients showed a changed in left ventricle ejection fraction of 3.5% relative to placebo, and left ventricular end-systolic volume of 8.5 ml over placebo.  When patients were asked to walk for six minutes, the JVS-100-treated patients were better than patients who had received the placebo.  Likewise, when patients were given the Minnesota Living with Heart Failure Questionnaire, the JVS-100-treat patients had a better score than those who had received the placebo.  Also, there were no unanticipated serious adverse events related to the drug reported for the study.

JVS-100 is a non-viral DNA plasmid gene therapy. Plasmids are small circles of DNA that are relatively easy to manipulate, grow and propagate in bacterial cells. In the case of the JV-100 treatment, the plasmid encodes a protein called stromal cell-derived factor 1 (SDF-1). SDF-1 is a naturally occurring signaling protein that recruits stem cells from bone marrow to the site of SDF-1 expression. SDF-1, therefore, acts as a stem cell recruitment factor that summons stem cells to the places where they are needed.

When JV-100 is delivered directly to a site of tissue injury, it induces the expression of SDF-1 protein into the local environment for a period of approximately three weeks. SDF-1 secretion creates a homing signal that recruits the body’s own stem cells to the site of injury to induce tissue repair and regeneration.

Juventas is developing JVS-100 into a treatment of advanced chronic cardiovascular disease, including heart failure and late stage peripheral artery disease.

These improvements in heart function are relatively modest.  Therefore, it is difficult to get too excited about these results.  Also, Alexey Bersenev, a umbilical cord stem cell researcher, noted that the primary end points (or goalposts) for this trial were not met, and that makes this an unsuccessful trial.  Despite this bad news, JV-100 does seem to be safe, and the theory seems sound, even if the results are more than a little underwhelming.

Experimental Drug Can Stimiulate the Regrowth of Damaged Tissues


Research at Case Western Reserve, in collaboration with scientists from UT Southwestern Medical Center has identified yet another stem cell-activating drug. In animal models, this drug has helped mice regrow damaged liver, colon, and bone marrow tissue. The experimental drug examined in these experiments might open new possibilities for regenerative medicine. If clinical trials show that this drug therapy works in humans, it might save the lives of critically ill people with liver or colon disease or even some cancers.

This study was published in the journal Science. Even this work is exciting, this research is in the early stages and more work is necessary for the drug can be tested in people.

“We are very excited,” said co-author Sanford Markowitz, professor of cancer genetics at Case Western Reserve’s School of Medicine. “We have developed a drug that acts like a vitamin for tissue stem cells, stimulating their ability to repair tissues more quickly,” he added. “The drug heals damage in multiple tissues, which suggests to us that it may have applications in treating many diseases.”

This new drug is called SW033291. SW033291 works by inhibiting an enzyme with the formidable name of 15-hydroxyprostaglandin dehydrogenase, which is mercifully shortened to 15-PGDH. This enzyme degrades regulatory molecules called “prostaglandins.” One of these prostaglandins, known as prostaglandin E2, stimulates stem cell growth and differentiation. Inhibition of 15-PGDH increases the concentrations of prostaglandin E2 and stimulates the growth of tissue stem cells, which promotes healing.

prostaglandin_e2

Markowitz and his colleagues first showed that SW033291 inactivated 15-PGDH in a test tube. When they fed the drug to cells, it also inhibited 15-PGDH. Finally, they gave the drug to lab animals and showed that even in a living body, SW033291 inhibited 15-PGDH.

Does the drug augment healing? To determine this, Markowitz and others subjected mice to lethal doses of radiation, followed by a partial bone marrow transplant. Some of the mice were given SW033291 plus the bone marrow transplant while others received only the transplant. The mice that received SW033291 survived, while the others died.

In other studies, mice that had lost large amounts of blood were given SW033291, and mice given SW033291 recovered normal blood counts six days faster than mice that did not get the treatment.

Mice with an inflammatory disease called ulcerative colitis were given SW033291 and the drug “healed virtually all the ulcers in the animals’ colons and prevented colitis symptoms,” said the study’s authors.

“In mice where two-thirds of their livers had been removed surgically, SW033291 accelerated regrowth of new liver nearly twice as fast as normally happens without medication.” Additionally, SW033291 produced no adverse side effects.

Researchers who were not involved with the work said the study showed promise, but urged a heavy dose of caution. For example, Dusko Illic, a stem cell expert at Kings College London, said: “The drug seems to be too good to be true. We would have to be sure that nothing else was wrong with any organ in the body,” because if there were cancer cells present, the treatment would likely cause tumor cells to grow along with other tissue.

However, Ilaria Bellantuono, an expert in stem cell science and skeletal ageing at the University of Sheffield, said a key part of the drug’s promise could be in helping cancer patients, if it is proven safe. The “treatment has the potential of boosting patents’ resilience and improving their response to cancer treatment,” said Bellantuono. “This study is a proof of concept in mice and more experimental work is needed to verify the long-term safety of such an approach but it surely shows promise.”

The author of this study said that the first people to receive the experimental treatment in clinical trials would likely be patients who are receiving bone marrow transplants, have ulcerative colitis, or are undergoing liver surgery.

Drug-Induced Regeneration in Adult Mice


What if you could take a pill to induce regeneration in your wounded body? Amphibians can lose a leg or tail and readily regenerate it spontaneously. Mammals, unfortunately, generally form scars over the injury site during the process of wound repair. However, a strain of mouse known as the MRL mouse strain is an exception because these mice have the ability to spontaneously regenerate and heal, and this animal is a model system for regeneration.

Ellen Heber Katz and her colleagues at the Wistar Institute in Philadelphia, Pennsylvania have examined the MRL mouse in some detail and have discovered that a protein called hypoxia-inducible factor 1α (HIF-1α) plays a central role in the regeneration ability of adult MRL mice. The HIF-1α protein is usually degraded when normal levels of oxygen are available. Degradation of HIF-1α is mediated by enzymes called PHDs, which stands for prolyl hydroxylases. The presence of oxygen provides the substrate for PHDs to modify HIF-1α so that the cell sees it as a protein that is marked for degradation. MRL mice seem to have a rather stable form of HIF-1α, which stimulates healing.

Heber Katz and her co-workers designed a drug that would inhibit PHDs and stabilize HIF-1α. Next they took their drug (1,4-dihydrophenonthrolin-4-one-3-carboxylic acid or 1,4-DPCA) and encased it in hydrogel that would slowly release it over a course of 4 to 10 days. Heber Katz and her fellow researchers then injected this drug-laced hydrogel beneath the skin of Swiss Webster mice, which do not show an ability to spontaneously regenerate. They discovered that their hydrogel increased stable expression of HIF-1α protein over a 5 day period. Then, when they subjected these mice to skin wounds by punching a hole in their ears, the Swiss-Webster mice showed regenerative wound healing. No stem cells were injected, but this drug-laced hydrogel increased the regenerative ability of these animals.

Thus, increased expression of the HIF-1α protein seems to provide a starting point for future studies on regeneration in mammals. This work in preliminary, but think of it – taking a pill or getting an injection of some hydrogel that increases your body’s healing ability many fold. Of course, this is far in the future, but the possibilities are remarkable.

MicroRNA Switches Improve Cell Purification


Stem cell-based therapies usually require the differentiation of stem cells into various cell types that are used for regenerative therapies. Such a strategy requires that the differentiated cells be purified from the rest of the cells. Typically, cell surface proteins are used as the means to distinguish cell types. Unfortunately, many undesired cell types may also share the same cell surface receptors, which will badly compromise the efficiency of cell purification.

Hirohide Saito from the Center for iPS Cell Research and Application (CiRA) at Kyoto University has designed a new way to isolate differentiated cells using microRNAs. This technique appears to be better than using cell surface proteins and it may revolutionize stem cell science.

Readers of this blog will recognize the term induced pluripotent stem cells or iPSCs, but for newer readers, I will provide a brief explanation of these cells. Induced pluripotent stem cells are made from mature, adult cells by means of genetic engineering and cell culture techniques. When the expression of four different genes (Oct4, Klf4, Sox2 and c-Myc) is forced in adult cells, a fraction of the cells de-differentiate and become like young, embryonic cells. When these cells are cultured ion special culture systems, they will aggregate and grow into an iPSC cell line. These cells have many, though not all, the features of embryonic stem cells, and they can, theoretically, differentiate into any adult cell type.

iPSCs are so popular in medical research because they are derived from a patient’s own body and they can be differentiate into any cell type. However, the protocols that are normally used to differentiate iPSCs lead to a mixed population of cells that are very heterogeneous, and the desired cell type has to be isolated from this mixture. Normally, antibodies that bind to surface receptors unique to the desired cell type are used for this purpose but in many cases such purification strategies are inefficient and the cell yield is rather poor. Also, these cell purification techniques have a tendency to damage cells.

New RNA-based procedures designed at CiRA may avoid these problems. Hirohide Saito and his colleagues designed tiny RNA molecules (microRNAs or miRNAs) that are designed to detect and sort live cells not by surface receptors, but by miRNAs. MicoRNAs are better markers of cell types and can improve purity levels. These “miRNA switches” as they are called, consist of synthetic mRNA sequences that include a recognition sequence for miRNA and an open reading frame (ORF) that codes a desired gene, such as a regulatory protein that emits fluorescence or promotes cell death. If the miRNA recognition sequence binds to miRNA expressed in the desired cells, the expression of the regulatory protein is suppressed, which helps distinguish one cell type from others that do not contain the miRNA and express the protein.

Senior Lecturer Yoshinori Yoshida, a heart muscle specialist who works with Professor Saito, immediately saw the potential of this technology. Dr. Yoshida has been studying how iPS cells can be used to combat cardiac diseases, but he has been stifled by unsatisfactory cell purification protocols. Heart muscle cells (cardiomyocytes) are especially difficult to purify because they do not possess unique cell surface proteins. So Professor Saito and Dr. Yoshida put their heads together to test the effectiveness of miRNA switches for isolating differentiated heart muscle cells from iPSCs.

First, they went to established heart muscle cell lines (which, by the way, are a colossal pain in the neck to deal with). They used these cells to define the miRNAs that are unique to cardiomyocytes. Then they designed several miRNA switches that contained sequences complementary to these miRNAs. After constructing the miRNA switches, they used them to isolated differentiated heart muscle cells from iPSCs.

The results were remarkable. Dr. Yoshida saw far better purification than he ever seen with standard methods. Furthermore, because this technology is RNA-based, it does not integrate into the genome and cause mutations. This could potentially make the cells eligible for clinical application.

Yoshida sees this tool as remarkably simple and something that can be used by stem cell researchers studying any organ. “It is just synthesizing RNA and transfecting them. It is not difficult,” he said. To prove this point, he and Saito used their miRNA switches to purify liver cells and pancreatic cells from iPSCs. This is significant, because neither of these cell types possess unique cell surface markers, but miRNA switches wre able to effectively purify them.

Intriguingly, the performance of different miRNA switches varied with the stages of cell development. This suggests that strategic selection of miRNAs could separate heart muscle cells that are at different developmental stages, which could also lead to even more homogeneous cell pools and potentially better cell therapy outcomes.

Saito believes that with further development, miRNA switches will be applicable to all cell types at all cell stages. “We want to make an active miRNA dictionary for each cell type, so that if we want to isolate this kind of cell type, we know how to use this kind of switch,” he said.

Epilepsy Reduces The Formation of New Neurons in the Brain


An ambitious, multidisciplinary project led by Amanda Sierra and Juan Manuel Encinas, Ikerbasque from the Achucarro centre (Achucarro Basque Center for Neuroscience) has discovered that epilepsy in a mouse model system reduces the production of new cells in the brain.

The hippocampus is a region of the brain involved in learning and memory and it is also the site of a robust neural stem cell population that generates new neurons. These hippocampal neural stem cells generate new neurons throughout the adult life of mammals. The cells generated by the hippocampal neural stem cells function in certain types of learning and memory and in responses to anxiety and stress.

Hippocampus

This new research by Sierra and Encinas has revealed that in epileptic mice, hippocampal neural stem cells stop generating new neurons and are turn into reactive astrocytes. Reactive astrocytes promote inflammation and alter communication between neurons. Could manipulation of neural stem cells provide new ways to treat epilepsy?

Reactive Astrocyte

Reactive Astrocyte

This work has recently been published in the journal Cell Stem Cell.

The results of this research also confirms previous work by the same group that showed that epilepsy, which causes hyperexcitation of neurons but does not cause convulsions, activates neural stem cells, which leads to their premature exhaustion. Thus the generation of new neurons in the hippocampus ends is chronically reduced.

Juan Manuel Encinas, the leader of this study, highlighted the fact that “this discovery has enabled us to gain a better understanding about how neural stem cells function. We have shown that in addition to generating neurons and astrocytes, neural stem cells in the adult hippocampus can generate reactive astrocytes following an epileptic seizure.”

Encinas and his colleagues carried out this work in experimental animals that were genetically engineered to be epileptic. However, this discovery has clear implications in clinical practice and in the quest to develop new therapies for epilepsy, since the generation of new neurons (neurogenesis) is a process that is negatively affected in epileptic seizures in the hippocampus. Encinas pointed out, “If we can manage to preserve the population of neural stem cells and their capacity to generate new neurons in humans, it may be possible to prevent the development of certain symptoms associated with epilepsy and very likely to mitigate the damage that is caused in the hippocampus.”

In this project, Encinas and his colleagues collaborated with research groups attached to institutions such as the Baylor College of Medicine in Houston (United States), the Université Catholique de Louvain (Belgium), the Achucarro centre itself, and the UPV/EHU’s Genetic Expression Service.