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.