Conditioning Stem Cells to Survive in the Heart


After a heart attack, the heart is a very inhospitable place for implanted stem cells. These cells have to deal with low oxygen levels, marauding white blood cells, toxins released from dead or nearly-dead cells, and other nasty things.

Getting cells to survive in this place is essential if the cells are going to provide any healing to he heart. Fortunately, a Chinese group has discovered that growing cells in inhospitable conditions before implantation greatly improves their survival. Now, this same group from Emory University School of Medicine in Atlanta, Georgia has shown that a small molecule can do the same thing.

This work, published in Current Stem Cell Research and Therapy, centers upon a pathway in cells controlled by a protein called the hypoxia-inducible factor or HIF. This protein regulates those genes that allow cells to withstand low-oxygen and other stressful conditions. HIF is composed of two parts: an oxygen-sensitive inducible HIF-1α subunit and a constitutive HIF-1β subunit. During nonstressful conditions, the alpha subunit is constantly being degraded after it is made because it is modified by a enzymes called prolyl hydroxylase (PHD) enzymes. In the presence of low oxygen conditions, PHD enzymes are inhibited and HIF-1α increases in concentration. The HIFα/β heterodimer forms and is stabilized, and translocates to the nucleus where it activates target genes.

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It turns out that small molecules can inhibit PHD enzymes and induce the low-oxygen status in cells without subjecting them to rigorous culture conditions. For example, dimethyloxalylglycine (DMOG) can inhibit PHD enzymes and produce in cells the types of responses normally observed under low-oxygen conditions.

In this paper, Ling Wei and colleagues cultured mesenchymal stem cells from bone marrow with or without 1 mM DMOG for 24 hours in complete culture medium before transplantation. These cells were then transplanted into the hearts of rats 30 minutes after those rats had suffered an experimentally-induced heart attack. They then measured the rates of cell death 24 hours after engraftment, and heart function, new blood vessel formation and infarct size 4 weeks later.

In DMOG-preconditioned bone marrow MSCs (DMOG-BMSCs), the expression of survival and blood-vessel-making factors were significantly increased. In comparison with control cells.  DMOG-BMSCs also survived better and enhanced the formation of new blood vessels in culture and when implanted into the heart of a living animal.
C to H , Angiogenesis was inspected using vWF staining (red) in heart sections from MI, C-BMSC and DMOG-BMSC groups 4 weeks after MI. Hoechst staining (blue) s hows the total cells. I. Summary of total tube length measured in experiments A and B. The t otal tube length in C- BMSC group was arbitrarily presented as 1. N = 3 independent measure ments. J , Summary of total vessel density in different groups of in vivo experiments. N = 8 animals in each group. * P <0.05 compared with C-BMSC group; # P <0.05 compared with MI control group.
C to H, Angiogenesis was inspected using vWF staining (red) in heart sections from MI, C-BMSC
and DMOG-BMSC groups 4 weeks after MI. Hoechst staining (blue) shows the total cells. I. Summary of total tube length measured in experiments A and B. The total tube length in C-BMSC group was arbitrarily presented as 1. N = 3 independent measurements. J, Summary of total vessel density in different groups of in vivo experiments. N = 8 animals in each group.
Transplantation of DMOG-BMSCs also reduced heart infarct size and promoted functional benefits of the cell therapy.
Effect of BMSCs transplantation on ischemia-induced infarct formation. Heart infarct area and scar formation were determined using Masson’s Trichrome staining 4 weeks after MI. A to C . Images of representative infarcted hearts from a MI control rat, a MI rat received C-BMSCs, and a MI rat received DMOG-BMSCs. D. Transplantation of BMSCs reduced heart infarction formation, the protective effects were significantly greater with transplantation of DMOG-BMSCs. N = 5 rats in each group. * P <0.05 compared with MI group; # P <0.05 compared with C-BMSC group.
Effect of BMSCs transplantation on ischemia-induced infarct formation. Heart infarct area and scar formation were determined using Masson’s
Trichrome staining 4 weeks after MI. A to C. Images of representative infarcted hearts from a MI control
rat, a MI rat received C-BMSCs, and a MI rat received DMOG-BMSCs. D. Transplantation of BMSCs
reduced heart infarction formation, the protective effects were significantly greater with transplantation of DMOG-BMSCs. N = 5 rats in each group.
Thus, this paper shows that targeting an oxygen sensing system in stem cells such as PHD enzymes (prolyl hydroxylase) provides a new promising pharmacological approach for enhanced survival of BMSCs.  This procedure also increases paracrine signaling, augments the regenerative activities of these cells, and, ultimately, and improves functional recovery of the heart as a result of cell transplantation therapy for the heart after a heart attack.  This is only a preclinical study, but the data is strong, and hopefully new clinical trials will bear this out.

Meta Study Shows that Mesenchymal Stem Cells Promote Healing in Animal Models of Stroke


Two scientists from my alma mater, UC Irvine, have examined experiments that treated stroke with bone marrow-derived stem cells. Their analysis has shown that infusions of these stem cells trigger repair mechanisms and limit inflammation in the brains of stroke patients.

UC Irvine neurologist Dr. Steven Cramer and biomedical engineer Weian Zhao identified 46 studies that examined the use of a specific type of bone marrow stem cells called mesenchymal stromal cells to treat stroke. Mesenchymal stromal cells are a type of multipotent adult stem cells that are found in many locations in the body. The best-known examples of mesenchymal stem cells are from bone marrow. When purified from whole bone marrow and used to treat stroke in animal models of stroke, Cramer and Zhao found that mesenchymal stromal cells (MSCs) were significantly better than control therapy in 44 of the 46 studies that were examined.

Further data culling of these studies showed that functional recovery from stroke were robust regardless of the MSC dosage or the time when MSCs were administered relative to the onset of the stroke, or the method of administration (whether introduced directly into the brain or injected via a blood vessel).

“Stroke remains a major cause of disability, and we are encouraged that the preclinical evidence shows [MSCs’] efficacy with ischemic stroke,” said Cramer, a professor of neurology and leading stroke expert. “MSCs are of particular interest because they come from bone marrow, which is readily available, and are relatively easy to culture. In addition, they already have demonstrated value when used to treat other human diseases.”

Another theme of these studies is that MSCs do not differentiate into brain-specific. MSCs have the capacity to differentiate into bone, cartilage and fat cells. “But they do their magic as an inducible pharmacy on wheels and as good immune system modulators, not as cells that directly replace lost brain parts,” he said.

In an earlier Cramer and Zhao examined the mechanism by which MSCs promote brain repair after stroke. These cells have the ability to home to the damages areas in the brain and release chemicals that stimulate healing. By releasing their cornucopia of healing-promoting molecules, MSCs orchestrate blood vessel creation to enhance circulation, the protection of moribund cells on the verge of death, and the growth of existing brain cells. Additionally, when MSCs reach the bloodstream, they settle in those parts of the body that control the immune system and they suppress the inflammatory response that can augment tissue damage. In this way, MSCs foster an environment more conducive to brain repair.

“We conclude that MSCs have consistently improved multiple outcome measures, with very large effect sizes, in a high number of animal studies and, therefore, that these findings should be the foundation of further studies on the use of MSCs in the treatment of ischemic stroke in humans,” said Cramer, who is also clinical director of the Sue & Bill Gross Stem Cell Research Center.

Stem Cells from Abdominal Fat Helps Fight Kidney Disease


Researchers from Chicago, Illinois have shown that a fatty fold of tissue within the abdomen contains a rich source of stem cells that can help heal diseased kidneys.

Scientists from the laboratory of Ashok K. Singh at Hospital of Cook County used a rat model of chronic kidney disease to examined the efficacy of these cells.

In past experiments, transplanted stem cells have failed to live very long in the body of the recipient. To solve this problem, Singh and his co-workers connected the a fatty fold of tissue located close to the kidney called the “omentum” to the kidney. The omentum is a wonderfully rich source of stem cells and by connecting the kidney to the omentum, Singh and his colleagues subjected the diseased kidney to a constant supply of stem cells.

Omentum

After 12 weeks of being connected to the kidney, the kidney showed significant signs of improvement.

The progression of chronic kidney disease was slowed due to this continuous migration of stem cells from the omentum to the diseased kidney. The influx of these stem cells seemed to direct healing of the kidney.

This experiment is significant in that it suggests that resident stem cells that facilitate healing of the kidney, but only when they are in contact with the tissue over a long period of time. Also, it implies that a supposedly useless organ that lies close to the kidney can be fused with the kidney to heal it with a patient’s own stem cells. This therapeutic strategy seems to be ideal for kidney patients.

Placenta-Based Stem Cells Increasing Healing of Damaged Tendons in Laboratory Animals


Pluristem Therapuetics, a regenerative therapy company based in Haifa, Israel, has used placenta-based stem cells to treat animal with tendon damage, and the results of this preclinical study were announced at a poster presentation at the American Academy of Orthopedic Surgeons’ (AAOS) annual meeting in New Orleans.

Dr. Scott Rodeo of New York’s Hospital for Special Surgery (HSS) is the principal investigator for this preclinical trial. His poster session showed placental-based stem cells that were grown in culture and applied to damaged tendons seemed to have an early beneficial effect on tendon healing. In this experiment, animal tendons were injured by treatments with the enzyme collagenase. This enzyme degrades tendon-specific molecules and generates tendon damage, which provides an excellent model for tendon damage in laboratory animals. These placenta-based cells are not rejected by the immune system and can also be efficiently expanded in culture. The potential for “off-the-shelf” use of these cells is attractive but additional preclinical studies are necessary to understand how these cells actually help heal damaged tendons and affect tendon repair.

“Although our findings should be considered preliminary, adherent stromal cells derived from human placenta appear promising as a readily available cell source to aid tendon healing and regeneration,” stated Dr. Rodeo.

“These detailed preclinical results, as well as the favorable top-line results we announced from our Phase I/II muscle injury study in January, both validate our strategy to pursue advanced clinical studies of our PLX cells for the sports and orthopedic market,” stated Pluristem CEO Zami Aberman.

Dr. Rodeo and his orthopedic research team at HSS studied the effects of PLX-PAD cells, which stands for PLacental eXpanded cells in a preclinical model of tendons around the knee that had sustained collagenase-induced injuries. Favorable results from the study were announced by Pluristem on August 14, 2013. Interestingly, Dr. Rodeo, the Principal Investigator for this study is Professor of Orthopedic Surgery at Weill Cornell Medical College; Co-Chief of the Sports Medicine and Shoulder Service at HSS; Associate Team Physician for the New York Giants Football Team; and Physician for the U.S.A. Olympic Swim Team.

Human Menstrual Blood Stem Cells Treat Premature Ovarian Failure in Mice


Premature ovarian failure (POF) or primary ovarian insufficiency is a condition characterized by loss of normal ovarian function before age 40. POF causes low levels of the hormone estrogen and irregular ovulation (release of eggs). POF causes infertility.

Some medical professional call POF premature menopause, even though these two conditions are not exactly the same. Women with POF may have irregular or occasional menstrual cycles for years and may even become pregnant. However, women with premature menopause cease having periods and can’t become pregnant.

The symptoms of POF are similar to those of menopause: irregular or skipped periods (amenorrhea), which may be present for years or may develop after a pregnancy or after stopping birth control pills; hot flashes, night sweats, vaginal dryness, irritability or difficulty concentrating, and decreased sexual desire.

In women with POF, infertility is very hard to treat, but restoring estrogen levels can avert many of the complications.

There are several causes of POF. Particular chromosomal defects such as Turner’s syndrome, in which a woman has only one X chromosome instead of the usual two, and fragile X syndrome, a major cause of intellectual disability can cause POF. Likewise, exposure to various toxins can also cause POF. Chemotherapy and radiation therapy are probably the most common causes of toxin-induced POF. Other toxins such as cigarette smoke, industrial chemicals, pesticides and viruses may also hasten POF. If the immune system mounts an immune response to ovarian tissue (autoimmune disease), then it might produce antibodies against the woman’s own ovarian tissue. Such antibodies will harm the egg-containing follicles and damage the egg. What triggers the immune response is unclear, but exposure to certain viruses is one possibility. Also various sundry unknown factors may also contribute to it.

There are no treatments for POF that restore the ovaries. For this reason a recent paper in the journal Stem Cells and Development represents a great advance in POF treatment.

Te Liu from the Shanghai Institute of Chinese Medicine and colleagues have used stem cells isolated from human menstrual blood to treat toxin-induced POF in mice.

Human endometrial stem cells exhibit stem cell properties in culture. These human endometrial stem cells are easily isolated from human menstrual blood. Other laboratories have even used them to treat heart conditions in clinical trials.

In this present study, Liu and colleagues treated female mice with the anti-cancer/anti-organ rejection drug cyclophosphamide. This drug pushed the mice into POF. Then one group of mice had human menstrual stem cells injected into their ovaries whereas another group received an injection of phosphate-buffered saline.

After 14 days, ovaries from those mice injected with human menstrual stem cells expressed higher levels of ovarian-specific proteins. Also, the blood levels of estrogen of the stem cell-injected mice were also higher. Postmortem examination also showed that the average ovarian weight of the stem cell-injected mice was much higher, as was the number of normal follicles. Follicles contain eggs surrounded with follicle cells and their absence is indicative of an ovary from a woman who is in menopause. That fact that the stem cell-treated POF mice had normal follicles and more of them suggests that the injected stem cells beefed up the supply of existing eggs and helped them survive and flourish.

These results suggest that these human menstrual stem cells, which are derived from the endometrium, can survive when introduced into a living organism and promote the regeneration of ovaries. There is no evidence that these cells differentiate into eggs, but instead they probably create an environment where the existing moribund eggs are rejuvenated and revitalized. This treatment for POF might be a viable option for human patients; all without destroying human embryos.

Transplanted Human Umbilical Cord Blood Cells Improved Long-Term Heart Muscle Structure and Function in Rats After a Heart Attack


Jianyi Zhang, from the University of Minnesota Health Science Center, in Minneapolis, Minnesota and his co-workers have shown that the transplantation of human umbilical cord blood cells into the rat hearts after a heart attack experience long-term effects that are not observed in the control animals that did not receive the stem cells. Furthermore, none of these laboratory animals required immunosuppressive therapy. The study is scheduled to be published in the journal Cell Transplantation.

“Myocardial infarction induced by coronary artery disease is one of the major causes of heart attack,” said Dr. Zhang. “Because of the loss of viable myocardium after an MI, the heart works under elevated wall stress, which results in progressive myocardial hypertrophy and left ventricular dilation that leads to heart failure. We investigated the long-term effects of stem cell therapy using human non-hematopoietic umbilical cord blood stem cells (nh-UCBCs). These cells have previously exhibited neuro-restorative effects in a rodent model of ischemic brain injury in terms of improved LV function and myocardial fiber structure, the three-dimensional architecture of which make the heart an efficient pump.”

According to Zhang and his co-authors, stem cell researchers have intently examined the ability of stem cells to regenerate and heal damaged heart tissue. Many laboratories all over the world have employed different types of stem cells, different animal models, and distinct modes of stem cell delivery into the heart tissue, and different stem cell doses. All of these studies have produced varying levels of improvement of left ventricular function. Zhang and others also note that, for the most part, the underlying mechanisms by which implanted stem cells improve heart function are “poorly understood and that the overall regeneration of heart muscle cells is modest at best.

In order to investigate the heart’s remodeling processes and to characterize the alterations in cardiac fiber architecture, Zhang’s team used diffusion tensor MRI (DTMRI), which has been previously used to study heart muscle fiber structure in both humans and animals. Most previous studies have concentrated on the short-term effects of umbilical cord blood cells (UCBCs) on damaged heart muscles. Fortunately, this study, which examined the long-term effects of UCBCs, not only demonstrated evidence of significantly improved heart function in treated rats, but also showed evidence of delay and prevention of myocardial fiber structural remodeling. Keep in mind that such alterations in heart muscle fiber structure could have resulted in heart failure.

When compared to the age-matched but untreated rat hearts that had suffered a heart attack, the regional heart muscle function of non-hematopoietic UCBC-treated hearts was significantly improved and the preserved myocardial fiber structure seems to have served as an “underlying mechanism for the observed function improvements.”

“Our data demonstrate that nh-UCBC treatment preserves myocardial fiber structure that supports the improved LV regional and chamber function,” concluded the researchers.

“This study provides evidence that UCBCs could be a potential therapy with long-term benefits for MI” said Dr. Amit N. Patel, director of cardiovascular regenerative medicine at the University of Utah and section editor for Cell Transplantation. “Preservation of the myocardial fiber structure is an important step towards finding an effective therapy for MIs”

See: Chen, Y.; Ye, L.; Zhong, J.; Li, X.; Yan, C.; Chandler, M. P.; Calvin, S.; Xiao, F.; Negia, M.; Low, W. C.; Zhang, J.; Yu, X. The Structural Basis of Functional Improvement in Response to Human Umbilical Cord Blood Stem Cell Transplantation . Cell Transplant. Appeared or available online: December 10, 2013.

Making Heart Muscle from Skeletal Muscle Stem Cells


Several experiments in animals and a few clinical trials in human patients have shown that implanting skeletal muscle cells isolated from muscle biopsies into the heart after a heart attack can help the heart to some degree, but the implanted skeletal muscle cells do not integrate into the existing heart muscle mass and the skeletal muscle cells do not differentiate into heart muscle cells.

Experiments like those mentioned above utilized muscle satellite cells. Muscle satellite cells are a resident stem cell population that respond to muscle damage and divide to form skeletal muscle cells form new muscle. Satellite cells are a perfect example of a unipotent stem cell, which is to say a cell that makes one type of terminally differentiated cell type.

Skeletal muscles, however, have another cell population called muscle-derived stem cells or MDSCs. MDSCs express an entirely different set of cell surface proteins than satellite cells, and have the capacity to differentiate into skeletal muscle, smooth muscle, bone, tendon, nerve, endothelial and hematopoietic cells. MDSCs grow well in culture, tolerate low oxygen conditions quite well, and show excellent regenerative potential.

Other laboratories have managed to culture MDSCs in collagen and produce beating heart muscle cells. Others have observed MDSCs forming a proper myocardium under certain conditions. Several studies have established the ability to MDSCs to treat laboratory animals that have suffered a heart attack. The most recent work from Sekiya and others has established that cell sheets made from MDSCs can reduce dilation of the left ventricle, increased capillary density, and promoted recovery without causing erratic heat beat patterns.

Despite their obvious efficacy. MDSCs remain difficult to isolate in high enough numbers to therapeutic purposes. None of the cell surface molecules sported by MDSCs are unique to those cells. Therefore, getting clean cultures of MDSCs remains a challenge. Still, these cells represent some of the best hopes for regenerative medicine in the heart. These cells do form heart muscle cells and heal ailing hearts. They can be grown in bioreactors to high numbers and can also be combined with engineered materials to shore up a damaged heart and mediate its regeneration. While the use of MDSCs is still in its infancy, the promise certainly is there.

Reversing Lung Diseases By Directing Stem Cell Differentiation


Lung diseases can scar the respiratory tissues necessary for oxygen exchange. Without proper oxygen exchange, our cells lack the means to make the energy they so desperately need, and they begin to shut down or even die. Lung diseases such as asthma, emphysema, chronic obstructive pulmonary disease and others can permanently diminish lung capacity, life expectancy and activity levels.

Fortunately, a preclinical study in laboratory animals has suggested a new strategy for treating lung diseases. Carla Kim and Joo-Hyeon Lee of the Stem Cell Research Program at Boston Children’s have described a new lung-specific pathway that is activated by lung injury and directs a resident stem cell population in the lung to proliferate and differentiate into lung-specific cell types.

When Kim and Lee enhanced this pathway in mice, they observed increase production of the cells that line the alveolar sacs where gas exchange occurs. Alveolar cells are irreversibly damaged in emphysema and pulmonary fibrosis.

Inhibition of this same pathway increased stem cell-mediated production of airway epithelial cells, which line the passages that conduct air to the alveolar sacs and are damaged in asthma and bronchiolitis obliterans.

For their experiments, Kim and Lee used a novel culture system called a 3D culture system that mimics the milieu of the lung. This culture system showed that a single bronchioalveolar stem cell could differentiate into both alveolar and bronchiolar epithelial cells. By adding a protein called TSP-1 (thrombospondin-1), the stem cells differentiated into alveolar cells.

Next, Kim and Lee utilized a mouse model of pulmonary fibrosis. However, when they cultured the small endothelial cells that line the many small blood vessels in the lung, which naturally produce TSP-1, and directly injected the culture fluid of these cells into the mice, the noticed these injections reverse the lung damage.

When they used lung endothelial cells that do not produce TSP-1 in 3D cultures, lung-specific stem cells produce more airway cells. in mice that were engineered to not express TSP-1, airway repair was enhanced after lung injury.

Lung Stem Cell Repair of Lung Damage

Lee explained his results in this way: “When the lung cells are injured, there seems to be a cross talk between the damaged cells, the lung endothelial cells and the stem cells.”

Kim added: “We think that lung endothelial cells produce a lot of repair factors besides TSP-1. We want to find all these molecules, which could provide additional therapeutic targets.”

Even though this work is preclinical in nature, it represents a remarkable way to address the lung damage that debilitates so many people. Hopefully this work is easily translatable to human patients and clinical trials will be in the future. Before that, more confirmation of the role of TSP-1 is required.

When Is the Best Time to Treat Heart Attack Patients With Stem Cells?


Several preclinical trials in laboratory animals and clinical trials have definitively demonstrated the efficacy of stem cell treatments after a heart attack. However, these same studies have left several question largely unresolved. For example, when is the best time to treat acute heart attack patients? What is the appropriate stem cell dose? What is the best way to administer these stem cells? Is it better to use a patient’s own stem cells or stem cells from someone else?

A recent clinical trial from Soochow University in Suzhou, China has addressed the question of when to treat heart attack patients. Published in the Life Sciences section of the journal Science China, Yi Huan Chen and Xiao Mei Teng and their colleagues in the laboratory of Zen Ya Shen administered bone marrow-derived mesenchymal stromal cells at different times after a heart attack. Their study also examined the effects of mesenchymal stem cells transplants at different times after a heart attack in Taihu Meishan pigs. This combination of preclinical and clinical studies makes this paper a very powerful piece of research indeed.

The results of the clinical trial came from 42 heart attack patients who were treated 3 hours after suffering a heart attack, or 1 day, 3 days, 2 weeks or 4 weeks after a heart attack. The patients were evaluated with echocardiogram to ascertain heart function and magnetic resonance imaging of the heart to determine the size of the heart scar, the thickness of the heart wall, and the amount of blood pumped per heart beat (stroke volume).

When the data were complied and analyzed, patients who received their stem cell transplants 2-4 weeks after their heart attacks fared better than the other groups. The heart function improved substantially and the size of the infarct shrank the most. 4 weeks was better than 2 weeks,

The animal studies showed very similar results.

Eight patients were selected to receive additional stem cell transplants. These patients showed even greater improvements in heart function (ejection fraction improved to an average of 51.9% s opposed to 39.3% for the controls).

These results show that 2-4 weeks constitutes the optimal window for stem cell transplantation. If the transplant is given too early, then the environment of he heart is simply too hostile to support the survival of the stem cells. However, if the transplant is performed too late, the heart has already experiences a large amount of cell death, and a stem cell treatment might be superfluous. Instead 2-4 weeks appears to be the “sweet spot” when the heart is hospitable enough to support the survival of the transplanted stem cells and benefit from their healing properties. Also, this paper shows that multiple stem cell transplants a two different times to convey additional benefits, and should be considered under certain conditions.

Cord Blood Stem Cells to Treat Acquired Hearing Loss


The Cord Blood Registry has announced the beginning of an FDA-regulated study at the Florida Hospital for Children in Orlando to investigate the potential of a child’s umbilical cord stem cells to treat acquired sensorineural hearing loss.

In the United States, about 15% of children suffer from low or high frequency hearing loss. Sensorineural hearing loss is the most common type of hearing loss, especially at high frequencies. Acquired sensorineural hearing loss results from damage to hair cells in the inner ear (cochlea) and can be caused by illness, medication, noise exposure, birth injury or head trauma. Because the ability to hear affects language development, hearing impairments can lead to poor academic and social development.

This particular study is a Phase I clinical trial, which will determine the safety and efficacy of using cord blood stem cells in children to improve inner ear function, and speech and language development.

In this study, the research group will follow 10 children who range in age from 6 weeks to 6 years, who have been diagnosed with acquired hearing loss for less than 18 months and who have had their own umbilical cord blood processed and stored.

Unfortunately, children who have a known genetic cause of deafness are ineligible for study participation. Patients will receive one intravenous infusion of their own umbilical cord blood stem cells. All patients will be tested at 1 month after the infusion, 6 months, and 1 year post-treatment.

As usual, this clinical trial is inspired by positive results in preclinical tests in laboratory animals.

Adult Stem Cells Used for Spinal Disc Repair


The Australian regenerative medicine company Mesoblast Limited announced the results of their 12-month clinical trial that examined the use of their “off-the-shelf” product to treat patients with disc-related low back pain.

This phase 2 clinical trial enrolled 100 patients with chronic moderate to severe “discogenic low back pain” and tested the ability of “mesenchymal precursor cells” to shore up degenerating intervertebral discs.

Intervertebral discs

Intervertebral discs sit between each vertebra and act as shock absorbers. Each disc consist of an outer layer called the “annulus fibrosus.” The annulus fibrosus consists of several layers of fibrocartilage. The annulus fibrosus surrounds an inner layer called the nucleus pulposus, which contains loose fibers suspended in a mucoprotein gel with the consistency of jelly. This jelly-like center distributes pressure evenly across the disc. These discs absorb the impact of the body’s daily activities and keep the two vertebrae separated. The development of a prolapsed disc results when the jelly in the nucleus pulposus is forced out of the doughnut/disc, which may put pressure on the nerve located near the disc.

Intervertebral structure

More than six million people in the United States alone deal with chronic back pain that has persisted for at least three months, and 3.5 million people are affected by moderate or severe degenerative intervertebral disc disease.

In this clinical trial, Mesoblast Limited injected their mesenchymal precursor cells (MPCs) into the degenerating intervertebral discs of patients suffering from moderate to severe back pain. When compared with a control group, patients who received the MPC injections used less pain killers, went through fewer surgeries and non-surgical interventions, and had greater disc stability as ascertained by X-rays. MPC injections also were well tolerated and produced few side effects.

This phase 2 clinical trial extends earlier observations by Mesoblast Limited on laboratory animals. In preclinical trials, purified MPCs increased the quality of the jelly content of the nucleus pulposus and improved disc structure in sheep.

This present study enrolled 100 patients at 13 different sites across Australia and the United States with early disc degeneration and randomly assigned the subjects to one of four groups: 1) those who received saline injections; 2) those who received hyaluronic acid injections; 3) those who received low-dose MPCs in hyaluronic acid; and 4) those who received high-dose injections of MPCs in hyaluronic acid.

All patients received their injections in an outpatient procedure, and are being evaluated for safety and efficacy to evaluate long-term treatment effects.

At 12 months, the key findings were improvement in chronic low back pain, function, and disc stability. Also, no safety concerns emerged as a result of the treatment.

As this trial proceeds, more data should be forthcoming.

Umbilical Cord Stem Cells Preserve Heart Function After a Heart Attack in Mice


A consortium of Portuguese scientists have conducted an extensive examination of the effects of mesenchymal stromal cells from umbilical cord on the heart of mice that have suffered a massive heart attack. Even more remarkable is that these workers used a proprietary technique to harvest, process, and prepare the umbilical cord stem cells in the hopes that this technique would give rise to a commercial product that will be tested in human clinical trials,

Human umbilical cord tissue-derived Mesenchymal Stromal Cells (MSCs) were obtained by means of a proprietary technology that was developed by a biomedical company called ECBio. Their product,, UCX®, consists of clean, high-quality, umbilical cord stem cells that are collected under Good Manufacturing Practices. The use of Good Manufacturing Practice means that UCX is potentially a clinical-grade product. Thus, this paper represents a preclinical evaluation of UCX.

This experiments in this paper used standard methods to give mice heart attacks that were later received injections of UCX into their heart muscle. The same UCX cells were used in experiments with cultured cells to determine their effects under more controlled conditions.

The mice that received the UCX injections into their heart muscles after suffering from a large heart attack showed preservation of heart function. Also, measurements of the numbers of dead cells in the heart muscle of heart-sick mice that did and did not receive injections of umbilical cord cells into their hearts showed that the umbilical cord stem cells preserved heart muscle cells and prevented them from dying. Additionally, the implanted umbilical cord MSCs induced the growth and formation of many small blood vessels in the infarcted area of the heart. This prevented the heart from undergoing remodeling (enlargement), and preserved heart structure and function.

When subjected to a battery of tests on cultured cells, UCX activated cardiac stem cells, which are the resident stem cell population in the heart. Implanted UCX cells activated the proliferation of cardiac stem cells and their differentiation into heart muscle cells. There was no evidence that umbilical cord MSCs differentiated into heart muscle cells and engrafted into the heart. Rather UCX seems to help the heart by means of paracrine mechanisms, which simply means that they secrete healing molecules in the heart and help the heart heal itself.

In conclusion, Diana Santos Nascimento, the lead author of this work, and her colleagues state that, “the method of UCX® extraction and subsequent processing has been recently adapted to advanced therapy medicinal product (ATMP) standards, as defined by the guideline on the minimum quality data for certification of ATMP. Given that our work constitutes a proof-of-principle for the cardioprotective effects UCX® exert in the context of MI, a future clinical usage of this off-the-shelf cellular product can be envisaged.”

Preclinical trials with larger animals should come next, and after that, hopefully, the first human clinical trials will begin.

Stimulus-Triggered Acquisition of Pluripotency Cells: Embryonic-Like Stem Cells Without Killing Embryos or Genetic Engineering


Embryonic stem cells have been the gold standard for pluripotent stem cells. Pluripotent means capable of differentiating into one of many cell types in the adult body. Ever since James Thomson isolated the first human embryonic stem cell lines in 1998, scientists have dreamed of using embryonic stem cells to treat diseases in human patients.

However, deriving human embryonic stem cell lines requires the destruction or molestation of a human embryo, the smallest, youngest, and most vulnerable member of our community. In 2006, Shinya Yamanaka and his colleges used genetic engineering techniques to make induced pluripotent stem (iPS) cells, which are very similar to embryonic stem cells in many ways. Unfortunately, the derivation of iPSCs introduces mutations into the cells.

Now, researchers from Brigham and Women’s Hospital (BWH), in Boston, in collaboration with the RIKEN Center for Developmental Biology in Japan, have demonstrated that any mature adult cell has the potential to be converted into the equivalent of an embryonic stem cell. Published in the January 30, 2014 issue of the journal Nature, this research team demonstrated in a preclinical model, a novel and unique way to reprogram cells. They called this phenomenon stimulus-triggered acquisition of pluripotency (STAP). Importantly, this process does not require the introduction of new outside DNA, which is required for the reprogramming process that produces iPSCs.

“It may not be necessary to create an embryo to acquire embryonic stem cells. Our research findings demonstrate that creation of an autologous pluripotent stem cell – a stem cell from an individual that has the potential to be used for a therapeutic purpose – without an embryo, is possible. The fate of adult cells can be drastically converted by exposing mature cells to an external stress or injury. This finding has the potential to reduce the need to utilize both embryonic stem cells and DNA-manipulated iPS cells,” said senior author Charles Vacanti, MD, chairman of the Department of Anesthesiology, Perioperative and Pain Medicine and Director of the Laboratory for Tissue Engineering and Regenerative Medicine at BWH and senior author of the study. “This study would not have been possible without the significant international collaboration between BWH and the RIKEN Center,” he added.

The inspiration for this research was an observation in plant cells – the ability of a plant callus, which is made by an injured plant, to grow into a new plant. These relatively dated observations led Vacanti and his collaborators to suggest that any mature adult cell, once differentiated into a specific cell type, could be reprogrammed and de-differentiated through a natural process that does not require inserting genetic material into the cells.

“Could simple injury cause mature, adult cells to turn into stem cells that could in turn develop into any cell type?” hypothesized the Vacanti brothers.

Vacanti and others used cultured, mature adult cells. After stressing the cells almost to the point of death by exposing them to various stressful environments including trauma, a low oxygen and acidic environments, researchers discovered that within a period of only a few days, the cells survived and recovered from the stressful stimulus by naturally reverting into a state that is equivalent to an embryonic stem cell. With the proper culture conditions, those embryonic-like stem cells were propagated and when exposed to external stimuli, they were then able to redifferentiate and mature into any type of cell and grow into any type of tissue.

To examine the growth potential of these STAP cells, Vacanti and his team used mature blood cells from mice that had been genetically engineered to glow green under a specific wavelength of light. They stressed these cells from the blood by exposing them to acid, and found that in the days following the stress, these cells reverted back to an embryonic stem cell-like state. These stem cells then began growing in spherical clusters (like plant callus tissue). The cell clusters were introduced into developing mouse embryos that came from mice that did not glow green. These embryos now contained a mixture of cells (a “chimera”). The implanted clusters were able to differentiate into green-glowing tissues that were distributed in all organs tested, confirming that the implanted cells are pluripotent.

Thus, external stress might activate unknown cellular functions that set mature adult cells free from their current commitment to a particular cell fate and permit them to revert to their naïve cell state.

“Our findings suggest that somehow, through part of a natural repair process, mature cells turn off some of the epigenetic controls that inhibit expression of certain nuclear genes that result in differentiation,” said Vacanti.

Of course, the next step is to explore this process in more sophisticated mammals, and, ultimately in humans.

“If we can work out the mechanisms by which differentiation states are maintained and lost, it could open up a wide range of possibilities for new research and applications using living cells. But for me the most interesting questions will be the ones that let us gain a deeper understanding of the basic principles at work in these phenomena,” said first author Haruko Obokata, PhD.

If human cells can be made into embryonic stem cells by a similar process, then someday, a simple skin biopsy or blood sample might provide the material to generate embryonic stem cells that are specific to each individual, without the need for genetic engineering or killing the smallest among us. This truly creates endless possibilities for therapeutic options.

New Analysis of Stem Cell Treatments for Spinal Cord Injury in Laboratory Animals


A host of preclinical studies have examined the ability of stem cells to improve the condition of laboratory animals that have suffered a spinal cord injury. While these studies vary in their size, design, and quality, there has been little cumulative analysis of the data generated by these studies.

Fortunately, there is a powerful analytical tool that can examine data from many studies and this type of analysis is called a “meta-analysis.” Meta-analyses use sophisticated statistical packages to systematically reassess a compilation of the data contained within these papers. Meta-analyses are exhausting, but potentially very useful. Such a meta-analysis is also very important because it provides researchers with an indication of what problems must be worked out before these treatments advance to human clinical trials and what aspects of the treatment work better than others.

A recent meta-analysis of stem cell therapy on animal models of spinal cord injury has been published by Ana Antonic, MSc, David Howells, Ph.D., and colleagues from the Florey Institute and the University of Melbourne, Australia, along with Malcolm MacLeod and colleagues from the University of Edinburgh, UK in the open access journal PLOS Biology.

The goal of regenerative spinal cord treatments is to use stem cells to replace dead cells within damaged areas of the spinal cord. Such treatments would be given to spinal cord injury patients in the hope of improving the ability to move and to feel below the site of the injury. Many experiments that utilize animal models of spinal cord injury have used stem cells to treat laboratory animals that have suffered spinal cord injury, but, unfortunately, these studies are limited in scale by size (as a result of financial considerations), practical and ethical considerations. Such limitations hamper each individual study’s statistical power to detect the true effects of the stem cell implantation. Also, these studies use different types of stem cells in their treatment scenarios, inject those cells differently induce spinal cord injuries differently, and test their animals for functional recovery differently.

To assess these studies, this new paper examined 156 published studies, all of which tested the effects of stem cell treatments on about 6,000 spinal cord-injured animals.

Overall, they found that stem cell treatment results in an average improvement of about 25 percent over the post-injury performance in both sensory (ability to feel) and motor (ability to move) outcomes. Unfortunately, the variation from one animal to another varied widely.

For sensory outcomes the degree of improvement tended to increase with the number of cells implanted. Such dose-responsive results tend to indicate that the improvements are actually due to the stem cells, and therefore, this stem cell-mediated effect represents a genuine biological effect.

The authors also measured the effects of bias. Simply put, if the experimenters knew which animals were treated and which were untreated, then they might be more likely to report improvements in the stem cell-treated animals. They also examined the way that the stem cells were cultured, the way that the spinal injury was generated and the way that outcomes were measured. In each case, important lessons were learned that should help inform and refine the design of future animal studies.

The meta-analysis also revealed some surprises that should provoke further investigations. For example, there was little evidence that female animals showed any beneficial sensory effects as a result of stem cell treatments. Also, the efficacy of the stem cell treatment seemed to not depend on whether immunosuppressive drugs were administered or not.

The authors conclude, “Extensive recent preclinical literature suggests that stem cell-based therapies may offer promise; however the impact of compromised internal validity and publication bias means that efficacy is likely to be somewhat lower than reported here.”

Even though human clinical trials are in the works, such trials will continue to be informed by preclinical studies on laboratory animals.

A Stem Cell-Based Therapy for Colon Cancer


Colorectal cancer is the third leading cause of death in the Western World. Like many other types of cancer, colorectal cancer spreads and is propagated by cancer stem cells. Therefore, understanding how to inhibit the growth of cancer stem cells provides a key to treating the cancer itself.

By inactivating a gene that drives stem cell renewal in cancer stem cells, scientists and surgeons at the Princess Margaret Cancer Centre in Toronto, Canada, have discovered a promising new approach to treating colorectal cancer.

John Dick, a senior scientist at the Princess Margaret Cancer Centre, said, “This is the first step toward clinically applying the principles of cancer stem cell biology to control cancer growth and advance the development of durable cures.”

In preclinical experiments with laboratory rodents, Dick and his team identified a gene called BMI-1 as a pivotal regulator of colon cancer stem cell proliferation. With this knowledge in hand, Dick’s laboratory dedicated many hours to finding small molecules that disarm BMI-1. Then Dick and his co-workers replicated human colorectal cancer in mice, and used their BMI-1-inhibiting small molecules to treat these cancer-stricken mice.

According to lead author of this work, Antonija Kreso: “Inhibiting a recognized regulator of self-renewal is an effective approach to control tumor growth, providing strong evidence for the clinical relevance of self-renewal as a biological process for therapeutic targeting.”

Dr. Dick explained: “When we blocked the BMI-1 pathway, the stem cells were unable to self-renew, which resulted in long-term and irreversible impairment of tumor growth. In other words, the cancer was permanently shut down.”

The clinical potential of this approach is significant, since it provides a viable treatment that specifically targets colon cancer. About 65% of all colorectal cancers have an activated BMI-1 pathway. Since physicians now have techniques for identifying the presence of BMI-1 and the tools to inhibit it, this strategy could translate into a clinical treatment that might radically transform the treatment of aggressive, advanced colorectal cancers. Such a treatment would be specific, personal, and specific. May the phase 1 trials begin soon!!!

Benefits of stem cells in treating MS declines with donor’s age


MS is a neurodegenerative disease characterized by inflammation and scar-like lesions throughout the central nervous system (CNS). There is no cure and no treatment eases the severe forms of MS. But previous studies on animals have shown that transplantation of mesenchymal stem cells (MSCs) holds promise as a therapy for all forms of MS (see Bai L, et al., Glia 2009 Aug 15;57(11):1192-203). The MSCs migrate to areas of damage, release trophic (cell growth) factors and exert protective effects on nerves and regulatory effects to inhibit T cell proliferation.

Several clinical trials examining the ability of fat-derived MSCs to treat MS patients have been conducted. Unfortunately, most of these studies are rather small and the results are all over the place. One study treated ten patients with MSCs injected intrathecally (just under the meninges that cover the brain and spinal cord) and the results were mixed; 6/10 improved, 3 stayed the same and one deteriorated. Another study treated ten patients with intravenous fat-derived MSCs and the patients showed symptomatic improvement, but when MRIs of the brain were examined, no improvements could be documented. A third study treated 15 people with intrathecal injections and IV administrations of MSCs, and some stabilized. A fourth study only examined 3 patients treated with a mixture of their own fat-derived MSCs and fat-derived MSCs from another person. In all three cases, their MRIs and symptoms improved. A fifth study used umbilical cord MSCs administered intravenously and the patient showed substantial improvement (for review see Tyndall, Pediatric Research 71(4):433-438).

These results are somewhat encouraging, but also somewhat underwhelming and clinical trials go. Why did some work and other not work as well? In order to understand why, researchers must understand the biologic changes and therapeutic effects of older donor stem cells. A new study appearing in the journal STEM CELLS Translational Medicine is the first to demonstrate that adipose-derived MSCs donated by older people are less effective than cells from their younger counterparts.

Fortunately, all the available MS-related clinical trials have confirmed the safety of autologous MSC therapy. As to the efficacy of these cells, however, it is unclear if MSCs derived from older donors have the same therapeutic potential as those from younger ones.

“Aging is known to have a negative impact on the regenerative capacity of most tissues, and human MSCs are susceptible to biologic aging including changes in differentiation potential, proliferation ability and gene expression. These age-related differences may affect the ability of older donor cells to migrate extensively, provide trophic support, persist long-term and promote repair mechanisms,” said Bruce Bunnell, Ph.D., of Tulane University’s Center for Stem Cell Research and Regenerative Medicine. He served as lead author of the study, conducted by a team composed of his colleagues at Tulane.

In their study, Bunnell and his colleagues induced an MS-like disease in laboratory mice called chronic experimental autoimmune encephalomyelitis (EAE). Then they treated them before disease onset with human adipose-derived MSCs derived from younger (less than 35 years) or older (over age 60) donors. The results corroborated previous studies that suggested that older donors are less effective than their younger counterparts.

“We found that, in vitro, the stem cells from the older donors failed to ameliorate the neurodegeneration associated with EAE. Mice treated with older donor cells had increased inflammation of the central nervous system, demyelination leading to an impairment in movement, cognition and other functions dependent on nerves, and a proliferation of splenocytes [white blood cells in the spleen], compared to the mice receiving cells from younger donors,” Dr. Bunnell noted.

In fact, the proliferation of T cells (immune cells that attack the myelin sheath in MS patients) in these mice indicated that older MSCs might actually stimulate the proliferation of the T cells, while younger stem cells inhibit T cell proliferation. T cells are a type of white blood cell in the body’s immune system that help fight off disease and harmful substances. When they attack our own tissues, they can cause diseases like MS.

As such, Dr. Bunnell said, “A decrease in T cell proliferation would result in a decreased number of T cells available to attack the CNS in the mice, which directly supports the results showing that the CNS damage and inflammation is less severe in the young MSC-treated mice than in the old MSC-treated mice.”

“This study in an animal model of MS is the first to demonstrate that fat-derived stem cells from older human donors have less therapeutic effectiveness than cells from young donors,” said Anthony Atala, M.D., editor of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine. “The results point to a potential need to evaluate cell therapy protocols for late-onset multiple sclerosis patients.”

Cardiac Stem Cells Offer New Hope for Treatment of Heart Failure


Scientists from the United Kingdom have, for the first time, highlighted the natural regenerative abilities of a group of stem cells that live in our hearts. This particular study shows that these cells are responsible for repairing and regenerating muscle tissue that has been damaged by a heart attack. Such damage to the heart can lead to heart failure.

There is a robust debate as to the regenerative capacity of cardiac stem cells (CSCs) in the hearts a adult human beings. While many scientists are convinced that CSCs in the hearts of newborns have good regenerative ability, many remain unconvinced that adult CSCs can do similar things (see Zaruba, M.M., et al., Circulation 121, 1992–2000 and Jesty, S.A., et al., Proc. Natl. Acad. Sci. USA 109, 13380–13385). Nevertheless, an earlier paper showed that when introduced into heart muscle after a heart attack, CSCs will regenerate the lost heart muscle and blood vessels lost in the infarct (see Beltrami, A.P., et al., Cell 114, 763–776). Resolving this disagreement requires a different type of experiment.

In this paper, Bernardo Nadal-Ginard and colleagues from the and his collaborators at the Stem Cell and Regenerative Biology Unit at the Liverpool John Moores University in Liverpool and his collaborators from Italy used a different way to affect the heart. When heart attacks are experimentally induced in the heart of rodents, the infarcts are large and they kill off large numbers of CSCs. Therefore, Nadal-Ginard and others induced severe diffuse damage of the heart muscle that also spared the CSCs. They gave the mice a large dose of a drug called isoproterenol, which acts as a “sympathomimetic.” This is confusing science talk that simply means that the drug speeds the heart rate to the point where the heart muscle exhausts itself and then starts to die off. This treatment, however, spares the CSCs (see Ellison, G.M., et al., J. Biol. Chem. 282, 11397–11409).

When the heart muscle was damaged, the CSCs differentiated into heart muscle cells and other heart-specific cells and repaired the damage in the heart. Also, the repairing cells were in the heart and were not the result of bone marrow stem cells that migrated to the bone marrow, thus putting to rest a controversy that has lasted for some years that CSCs are the result of bone marrow stem cells that migrate to the heart.

Elimination of CSCs prevents heart repair after heart damage. If, however, these heart-based stem cells are replaced after damage, the heart repairs itself and the heart recovers its function, anatomical integrity, and cellular structure.

In other experiments, removal of cardiac stem cells (CSCs) and re-injection after a heart attack shows that the CSCs can home in and repair the damaged heart.

c-kit CSCs repair heart

Since Nadal-Ginard showed that CSCs have a capacity to home to the damaged heart, less invasive treatments might be possible and that these treatments might even prevent heart failure after a heart attack in the future.

In a healthy heart, the quantity of CSCs is sufficient to repair heart muscle tissue. However, once the heart is damaged many of the CSCs are also damaged and cannot multiply or produce new muscle tissue. In these cases it could be possible to replace damaged CSCs with new ones that have been grown in the laboratory and administered intravenously.,

These new approaches involved maintaining or increasing the activity of CSCs in order to renew heart muscle and replace old, damaged cells. This new strategy will only require intravenous administration of CSCs and not require open heart procedures that require such a long time to recover.

These findings are very promising. The nest step is a clinical trial, which is due to start early 2014 and is aimed at assessing the safety and effectiveness of CSCs for preventing and treating heart failure in humans.

GERON’S IND FOR SPINAL CORD INJURY PLACED ON HOLD


Geron Corporation has made a cell line called GRNOPC1 from embryonic stem cells. GRNOPC1 is an “oligodendrocyte precursor cell” or OPC line. Before you blow a gasket at the sight of such a long-winded description, just remember that nerves are like wires and wires need insulation.  OPCs are the cells that make the insulation.  During spinal cord injury, the insulation dies off and it causes nerves to malfunction.

In collaboration with Hans Keirstead at UC Irvine, Geron developed a protocol for the administration of GRNOPC1 cells to animals with acute spinal cord injuries. His protocol showed that the OPCs were safe (no tumors were seen, even after one year) and somewhat effective. Some scientists were skeptical, since the mice had somewhat less severe spinal cord injuries.  Nevertheless, Geron was granted an Investigational New Drug Application from the FDA to conduct a Phase I trial with their OPC cell line.

They apparently, however, have bit a bit of a snag. Here is a press release from Geron Corporation.

Geron Corporation today announced that its IND (Investigational New Drug application) for GRNOPC1, a cell therapy for neurologically complete, subacute spinal cord injury, has been placed on clinical hold by the FDA pending the agency’s review of new nonclinical animal study data submitted by the company. A clinical hold is an order that the FDA issues to a sponsor to delay a proposed trial or to suspend an ongoing trial.

Since filing the IND, Geron has been undertaking studies to enable dose escalation of its spinal cord injury product, and has been investigating application of the product to other neurodegenerative diseases. The company has also been performing additional product characterization and conducting further animal studies. Data from this work has been submitted to the FDA. Geron will work closely with the FDA to facilitate their review of the new data and to release the clinical hold. No patients have yet been treated in this study.

From the sound of it, this hold is merely an administrative procedure that the FDA routinely undergoes when presented with new data.  However, if the new data is completely consonant with previous findings, why would there be a hold? We simply do not know at this time.  It is entirely possible that nothing is amiss, and this is merely FDA policy.  However, it is also possible that Geron’s new product does not behave exactly as they thought.

The development of the first cholesterol-lowering drug (lovastatin) experienced a slow-down when a related product being developed in Japan caused cancer in dogs. Roy Vagelos, president of Merck at the time, contacted the FDA and suspended all clinical trials. Further testing by Merck showed that this was an anomaly, and extensive clinical use has vindicated this finding. Maybe this is a similar situation for Geron’s OPC line?  Only time will tell.