Preconditioning Stem Cells from Fat with Viagra Improves their Therapeutic Efficacy

Rackesh Kukreja is professor of internal medicine, biochemistry, and molecular biology, physiology and biophysics, and scientific director of the Virginia Commonwealth University Pauley Heart Center. In his laboratory, Nicholas Hoke and other co-workers have discovered a way to improve the therapeutic capacity of fat-derived mesenchymal stem cells (MSCs).

MSCs from fat can be transformed into heart muscle cells in the laboratory (see Rangappa et al., Ann Thorac Surg. 2003; 75(3):775-9). While these cells will beat in culture, it is unclear if these cells have the calcium ion-handling machinery that allows them to synchronously beat together. When implanted into the hearts of laboratory animals that have suffered heart attacks, stem cells from brown fat shrink the infarcted area and improve the function of the left ventricle. This improvement seems to occur by means of replacing dead heart muscle cells (see Yamada, et al., Biochem Biophys Res Commun. 2006; 342(2):662-70). Several experiments have established that MSCs from fat tissue can improve the function of a rodent’s heart after a heart attack. Sheets of fat-derived MSCS (see Miyahara et al., Nat Med. 2006; 12(4):459-65), cord blood MSCs co-cultured with brown fat stem cells (see Yamada, et al., Biochem Biophys Res Commun. 2007; 353(1):182-8), or even MSCs derived from white adipose tissue (see Schenke-Layland K, et al., J Surg Res; 153(2):217-23; Mazo M, et al., Eur J Heart Fail. 2008; 10(5):454-62; Valina C, et al., Eur Heart J. 2007; 28(21):2667-77) all improved heart function after a heart, the fat-derived MSCs from white fat seemed to do so by enhancing blood vessels formation in the infarcted heart. Other studies showed that white fat derived MSCs did not survive well in the heart after a heart attack (van der Bogt, et al., Transplantation. 2009; 87(5):642-52). Other studies showed that fat-derived MSCs could be converted into heart muscle cells (Gwak, et al., Cell Biochem Funct. 2009; 27(3):148-54), and transplantation of these cells could improve heart function after a heart attack (Okura H, et al., Tissue Eng Part C Methods. 2010; 16(3):417-25). Therefore, fat-derived MSCs have the potential to help an ailing heart, but can we tweek them so that they can survive within the hostile environment of a heart that has just had a heart attack?

Into this gap steps Rackesh Kukreja, Nicholas Hoke and co-workers and their experiments on fat-derived MSCs. They soaked fat-derived MSCs in sildenafil (Viagra) before they transplanted them into rodent hearts after a heart attack. Before you snicker about this, sildenafil is an inhibitor of enzymes called “phosphodiesterases.” Phosphodiesterases degrade signaling molecules like cyclic AMP and cyclic GMP. Both of these molecules are made when cells receive messages from other cells, and the concentration of these molecules inside can determine if a cell survives under certain conditions or dies. Therefore, by treating the cells with sildenafil (Viagra), they increased the intracellular concentration of these signaling molecules.

The results were remarkable. When they injected these preconditioned cells directly into the heart muscle of mice that had experienced a heart attack, the preconditioned cells released more growth factors, survived better than non-conditioned fat-derived MSCs, and also helped repair the heart much more effectively. There was decreased cell death in the hearts treated with the preconditioned MSCs, greater density of new blood vessels, and far less scarring of the heart.
These results demonstrate that preconditioning MSCs from fat with drugs like Viagra is a powerfully simple and novel approach to improve stem cell therapy following a heart attack.

Integrin α6β4 identifies an adult lung stem cell population with regenerative potential

Can damaged lung tissue regenerate? If so, which cells contribute to this regeneration? Can we isolate these regenerative cells and make them available to people with failing lungs?

These are all pointed questions, and associate professor of medicine at the University of San Francisco, Thiennu H. Vu, has published a recent paper in the Journal of Clinical Investigation that partially answers these questions.

The lung consists of a large quantity of tubes that conduct the air to the bloodstream. These tubes, the trachea, bronchi, bronchioles and terminal bronchioles, constitute the “conducting zone” of the lungs. They serve to deliver the air from outside our bodies to the bloodstream. The actual site of gas exchange or “respiratory zone” occurs at the “alveoli.” The terminal bronchioles end in an inflation that resembles a tiny sac. This sac, the alveolus, is very thin; one cell thick.

The cells that compose the alveolus are called alveolar epithelial cells (AECs). There are two types of AECs: flat “type I pneumocytes,” which typically are unable to divide and die off they are damaged by toxins, and “type II pneumocytes,” also known as “great alveolar cells” or “septal cells.”

Type II pneumocytes are usually found near the junctions between alveoli and the septae that separate the alveoli. It is thought that type II cells can divide and replace type I cells if the type I cells are destroyed. Type II cells also secrete large quantities of “surfactant” which is a chemical that keeps the alveolar surfaces from sticking together as they expand and contract. Are type II pneumocytes the primary healing cell in the lung? Vu’s group set out to address this question.

Vu and her co-workers had an indication that mice that lack a particular surface molecule called “integrin beta4” could not repair their lungs after lung damage. Integrins are cell adhesion molecules that help cells stick to the substratum. If we think of lung cells as having a head (the apical surface), and a foot (the basal surface), the foot part of the cell stands on a foundation and this foundation in lungs is something called the “basement membrane.”

Basement membranes are common to other types of cells, but basement membranes in the lung are rich in a protein called “laminin,” and the beta4 integrin, with help from another integrin subunit called alpha6, binds tightly to laminin and keeps the lung cells lock to the foundational basement membrane.

Since the cells that contained alpha6/beta4 on their surfaces seemed to the cells responsible for regenerating the lung after the lung was damaged, Vu and her colleagues stained lung tissue with antibodies against the beta4 integrin. What they discovered surprised them: The beta4-expressing cells did NOT overlap with those cells that made surfactant (type II cells). Furthermore, when they tried to correlate the presence of the beta4 integrin with the available lung cell types (type I AECs, ciliated bronchial cells, type II AECs, and Clara cells), they were not able to show that these beta4 cells corresponded to any known lung cell type.

Next, Vu and others cultured lung cells in artificial media and the beta4 integrin-containing cells grew extremely well, but the other lung cells failed to grow. The growing beta4-positive cells also proved to be a mixed population and had the beta4 integrin in common, but little else.

The next experiment utilized a culture system that Vu’s lab helped develop whereby extirpated lung tissues are used to grow mini-lung-like organs when transplanted into a “nude” mouse (a mouse whose immune system does not work properly). By using a nude mouse, the implanted cells will form the mini-lung without the mouse’s immune system destroying it. By using their mini-lung growing system, Vu and her colleagues were able to grow the mini-lungs effectively if they used whole, macerated lung tissue. The growing lungs went through the various embryonic stages of lung development, thus showing that this assay is an excellent way to study lung development. Next they tried to grow the mini-lungs by using only integrin beta4-containing lung cells plus some embryonic cells. The beta4-positive cells grew into mini-lungs and formed a wide variety of lung-specific cell types. The integrin beta4-containing cells also directed the embryonic epithelial cells to form proper sac-like alveoli. This assay definitively showed that the beta4-positive cells could form type I and type II pneumocytes.

Finally, they injured the lungs of mice with a drug called bleomycin and looked at the cells in the lungs to see if the quantity of beta4-containing cells increased. The results were crystal clear; the beta4-positive cells increased many fold. Then they asked if the type II pneumocytes were dividing in the damaged lungs. They used genetically engineered mice that would express green fluorescent protein in their type II pneumocytes. Then they injured the lungs of these mice and asked if the type II cells increased their numbers. The answer was a clear NO. The regeneration that created new type II pneumocytes created cells that did not express green fluorescent protein, which means that the new type II cells were made from cells that did not originally express green fluorescent protein. Therefore, the beta4-positive cells were the cells regenerating the lung and not the type II cells. The type II cells that were dividing had been derived from the beta4-positive cells.

Vu and her colleagues end this paper with this modest understatement: “Understanding the determinants of β4+ AEC population size and how these cells expand, self-organize, and differentiate along particular lineages should provide further insights into the processes of lung repair, the foundation for better therapeutics.”

I’ll say. If these cells can be found and characterized in humans, they could revolutionize lung treatments. That would be a revolutionary treatment.

Positive Results From Phase 2 Trial Of Mesoblast’s Adult Stem Cell Therapy

Mesoblast announced positive Phase 2 heart failure trial results of its off-the-shelf, adult stem cell product Revascor after all patients had completed a minimum follow-up of 12 months, and a mean follow-up of 22 months. The Phase 2 trial results were presented at the American Heart Association annual meeting in Orlando, Florida, by independent principal investigator Dr Emerson C. Perin, Director of Research in Cardiovascular Medicine and Medical Director, Stem Cell Center, Texas Heart Institute in Houston.

Mesenchymal Precursor Cells or MPCs are bone marrow stem cells that have none of the markers expressed by mature mesenchymal stem cells (MSCs), but they are the stem cells population that gives rise to mesenchymal stem cells. Therefore, they have the advantages of MSCs – such as they are not recognized by the immune system, but because they are not mature MSCs, they can differentiate into a far wider variety of cell types than mature MSCs.

MPC treatment in this trial pooled data from patients that received all different doses and these pooled data showed that patients who had received MPC treatments had a significant reduction in cardiac mortality. Furthermore, at the highest dose, the MPCs completely prevented heart failure hospitalization events. Mesoblast expects that these outcomes will be central to the primary endpoint of a Revascor Phase 3 trial for product regulatory approval by the United States Food and Drug Administration (FDA).

This phase II trial used a randomized, placebo-controlled 60-patient Phase 2 trial that compared the safety and efficacy of three doses of Revascor in addition to maximal approved therapies versus maximal therapies alone in patients with moderate-to-severe congestive heart failure (CHF) defined by New York Heart Association (NYHA) class II or III status and ejection fraction below 40%. The trial enrolled both ischemic and non-ischemic heart failure patients. Heart failure patients with this degree of severity are known to have a high cardiac mortality over a 12-24 month period despite being on maximal approved drug and device therapies.

Treatment with MPCs was well-tolerated. Over a 22-month mean follow-up period, only 1/45 (2%) patients who received a single injection of Revascor died of cardiac causes compared with 3/15 (20%) of the control group (p=0.02). In addition, MPC treatment significantly delayed the time to a first Major Adverse Cardiac Event, MACE, a composite of cardiac death, heart attack or revascularization procedures (p=0.036), and reduced the overall risk for MACE by 78% (p=0.011). Over a mean follow-up of 18 months, 0/15 patients who received the highest dose of MPC (150M) had been hospitalized for heart failure or had died. In contrast, 3/15 (20%) controls and 6/30 (20%) patients who received low (25M) or mid (75M) doses of MPC had either been hospitalized with heart failure or had died. This clinical improvement associated with the 150M dose was accompanied by evidence of cardiac remodeling (reduction in left ventricular end systolic volumes compared with controls at 6 months, p=0.015) and improved functional heart capacity (gain of 52.6 meters over 6 minutes’ walk compared with controls at 12 months, p=0.06).

After 12 months, 40% of all treated patients had reverted to class I NYHA status compared with 14% of all controls, and this effect remained when patients were matched for the presence of class II status at baseline. The group who received the 25M MPC dose showed a significant 8.9 point improvement in ejection fraction over controls at 3 months (p=0.008), with a sustained but less pronounced effect over 12 months. In contrast, the group who received 150M MPC did not show improved ejection fraction, suggesting that the positive effects of this dose on clinical outcomes, remodeling, and functional capacity may be due to other mechanisms such as anti-fibrosis.

Dr. Perin stated: “These clinical findings are the first using any cell therapy in heart failure patients to show a concordant positive effect on clinical outcomes, cardiac remodeling, and functional capacity, the three key parameters in congestive heart failure. Together, they indicate that a single 150 million dose of Revascor may significantly reduce both heart failure hospitalizations and death in these very sick patients who have such a poor prognosis despite maximal existing therapies. Based on their defined product characterization, batch to batch consistency, immediate availability, and lack of clinically relevant immunogenicity, MPCs appear to be an ideal cell type to provide a new level of patient care in congestive heart failure. We look forward to progressing the Revascor clinical program into Phase 3.”

Revascor is being jointly developed by Mesoblast and its strategic alliance partner, Teva Pharmaceutical Industries Ltd. Teva’s Corporate Vice President Global Branded Products, Kevin Buchi, said: “These independently-reviewed results serve to reinforce Teva’s commitment to its strategic investment in Mesoblast’s adult stem cell technology and to our continued support for the clinical development of Revascor.”

Mesoblast Chief Executive, Professor Silviu Itescu, said, “Together with our partners at Teva, we are deeply committed to bringing to market an effective cell therapy product to reduce recurrent hospitalization episodes and risk of death in patients with progressive heart failure. The exciting results presented at the American Heart Association meeting reinforce the strength of our technology and emphasize the need to maintain a rapid development path in order to make this product available for the many patients suffering with heart failure.”

New Heart Cells Increase By 30 Percent After Stem Cell Infusion

Chronic ischemic heart disease results from the partial blockage of blood flow to the heart. It can result in damage to the heart, and symptoms that consist of shooting pain in the chest called “angina.” Fortunately, there are good, animal models of chronic ischemic heart disease and better ways to treat this disease are being investigated. A presentation at the American Heart Association annual meeting has shown that new heart cells can be produced in animals that have been given infusions of stem cells derived from cardiac biopsies or “cardiospheres.”

Research conducted at the University at Buffalo School of Medicine and Biomedical Sciences has demonstrated that the hearts of animals with chronic ischemic heart disease experience a 30 percent increase in healthy heart muscle cells within one month after receiving cardiosphere-derived cells (or CDCs). This finding is contradicts conventional medical wisdom which avers that heart cells are terminally differentiated and thus, are unable to divide.

Ischemic heart disease results from narrowing of coronary arteries and prior heart attacks are the most common cause of heart failure. Other investigators have largely focused on regenerating muscle in scarred tissue, but this UB group has shown that cardiac repair can be achieved by infusing CDCs slowly into coronary arteries of the diseased as well as normal areas of the heart. Study co-author John M. Canty Jr., MD, the Albert and Elizabeth Rekate Professor of Medicine in the UB medical school and UB’s chief of cardiovascular medicine explains: “Whereas most research has focused upon irreversible damage and scarring following a heart attack, we have shown that a single CDC infusion is capable of improving heart function in areas of the heart that are viable but not functioning normally.” Particular areas of heart dysfunction even their there is no fibrotic scarring are common in patients with heart failure from coronary artery disease. Heart failure results from “remodeling” in response to a heart attack, in which the heart enlarges to adjust to the loss of heart muscle. Another consequence of a heart attack and periods of inadequate blood flow to the heart muscle is so-called hibernating myocardium, in which segments of heart muscle exhibit abnormalities of contractile function. Canty commented further: “The rationale for our approach is somewhat analogous to planting seeds in fertile soil versus trying to grow plants in sand.

Gen Suzuki, MD, research assistant professor of medicine in the UB medical school and lead author on the research, noted: “We have shown that cells derived from heart biopsies can be expanded outside of the body and slowly infused back into the coronary arteries of animals with chronic dysfunction from restricted blood flow or hibernating myocardium. The new cardiac muscle cells are small and function more normally than diseased large, hypertrophied myocytes.”

Canty also noted that infusing stem cell formulations directly into coronary arteries also delivers the cells throughout the heart and is much simpler than injecting cells directly into heart muscle which requires equipment that is not widely available.

The research currently is in a preclinical phase but the UB researchers expect that translation to determine effectiveness in patients could take place within two to three years or possibly even sooner.

Stem cell therapy helps kidney transplant patients

Organ transplants usually require transplant patients to take anti-rejection drugs that suppress the immune system of the patients. Transplant patients must take these drugs for the rest of their lives to prevent the patient’s immune system from attacking and damaging the transplanted organ. These anti-rejection drugs, however, have serious side effects. These drugs can prevent the immune system from fighting infections, and this means that viruses can attack the pancreas, thus leading to diabetes, and tumors can pop up without being restricted by the immune system.  Anti-rejection drugs include such drugs as glucocorticoids, cyclophosphamide (very potent), folic acid analogues like methotrexate, purine analogues like azathioprine, and mercaptopurine, cyclosporin, tacrolimus, and sirolimus, and a cytotoxic antibiotic like dactinomycin.

In order to decrease the need for immunosuppressive drugs, a new technique is being developed that adds adult stem cells plus an initial anti-rejection drug treatment seems to allow the immune systems of kidney transplant patients to accept their transplanted organ without the need for lifelong drug therapy.

The procedure goes something like this: First the patient receives the new kidney, followed by a targeted dose of radiation to weaken their immune system and make some room for new immune cells. Next comes a bone marrow transplant from the same donor who donated the kidney. These bone marrow stem cells mix with the patient’s cells and the transplant patient now has an immune system that is a mosaic of their own original immune system and that of the donor’s. This new immune system views the transplanted kidney as a “perfect match.” Thus far, 8 out of 12 patients who have received the new kidney plus the additional bone marrow transplant are progressing without any need for anti-rejection drugs. Adult bone marrow stem cells provide the life-saving support needed for organ transplants.

More on Geron Leaving the Embryonic Stem Cell Business

According to the Washington Post, “Geron Corp., a pioneer in stem cell research that has been testing a spinal cord injury treatment, said late Monday that it’s halting development of its stem cell programs to conserve funds. It is seeking partners to take on the programs’ assets and is laying off much of its staff…In a statement, the company said the decision to narrow its focus “was made after a strategic review of the costs, … timelines and clinical, manufacturing and regulatory complexities associated with the company’s research and clinical-stage assets.”

Bio-ethicist Wesley Smith sees something potentially nefarious at work. Geron was in the midst of a Phase I clinical trial for its embryonic stem cell-derived oligodendrocyte precursor cell line GRNOPC1. Embryonic stem cells (ESCs) are made from human blastocyst-stage embryos. The derivation of ESC lines requires the dis-assembly, and consequent death of the embryo, which is a human person in the very early stages of development. The GRNOPC1 cell line was made from ESC line H1, which was originally made in the laboratory of James Thomson at the University of Wisconsin when he made the first human ESC lines. H1 was originally called WA01.

The cell line GRNOPC1 was made by differentiating H1 cells into oligodendrocyte progenitor cells (OPCs). OPCs are stem cells found in the central nervous system. They divide to form oligodendrocytes, which wrap around the axons of neurons and help them conduct their nerve impulses much faster than they normally would. Because oligodendrocytes take a beating during spinal cord injury, replacing them can potentially help spinal cord injury patients recover some function. Transplanted GRNOPC1 cells have been shown to improve the function of mice that have suffered spinal cord injuries. Geron has also done quite of bit of work to establish that GRNOPC1 cells are safe, at least in mice.

According the Geron’s web site, Geron worked with collaborators at various universities to demonstrate that GRNOPC1 improved movement in spinal cord-injured animals if they were implanted seven days after the spinal cord injury. When Geron scientists examined the spinal cords of the animals that had received the GRNOPC1 cells, they found that the transplanted OPCs had engrafted or became a part of the spinal cord and were functioning as expected. They published these data in May 2005 in the Journal of Neuroscience. Geron and it collaborators also implanted GRNOPC1 cells into spinal cord injured animals nine months after injury. Tissue examinations of these laboratory animals showed that the site of spinal cord injury was filled with GRNOPC1 cells and properly myelinated rat axons that were crossing the lesion. This is something that axons do not do after spinal cord injury, because the inflammation in the spinal cord kills all neurons in the area and axons that traverse the damaged area. Neurons may not be anywhere near the area of spinal cord damage, but if their axons (the portion of the neuron that directs nerve impulses away from the cell body of the neuron) extend through the area, the axons are severed and the neuron retracts its axons. The target that was receiving nerve inputs from the axon now lacks any input from the nervous system and if that target is a skeletal muscle, which is the target for motor neurons, the muscle becomes incapable of contracting.

Geron also notes on its website that all preclinical studies performed in animals provided the rationale for the use of GRNOPC1 in treating spinal injuries in human clinical trials. Extensive safety testing also satisfied the Food and Drug Administration that they were safe for use in humans. The FDA, therefore, gave Geron the green light to test their GRNOPC1 cell line in human patients who had recently suffered spinal cord injuries.

However, Geron is now leaving the very field it pioneered. This is being reported as a calculated business move that is due to the gargantuan financial investment required to bring embryonic stem cells into the world of everyday medicine. Late Monday this week (November 14, 2011), the company said it would cease all studies of stem cell-based treatments for spinal cord injury. This treatment was the first ever embryonic stem cell trial approved in the U.S. Geron, a Menlo Park, California, company has long been viewed as the undisputed leader in stem cell therapies. This leadership role is due to patents Geron holds on technology used to grow, manipulate and inject stem cells into the human body. In fact, Geron helped finance researchers at the University of Wisconsin who first isolated human embryonic stem cells in 1998, allowing the cells to be grown in the laboratory.

Wesley Smith smells a potential rat. He asks the following questions: “That being so, Geron and the media have an obligation to explain the why of this story in some detail and without spin. Was it the recent European ruling banning the patenting of embryonic stem cell products (about which I wrote) a factor? Was its human trial a disappointment? If it is out of money, why aren’t venture capitalists more willing to invest more in the field if it is so promising? I am sure you all have questions of your own.” These are all good questions, but it is entirely possible that Geron, having already sunk 25 million dollars into this venture and seeing that their product was still decades away from coming to the market, they decided to cut their losses and sell it to someone else. Geron has some anti-cancer drugs in the clinical trial pipeline that are doing very well in their clinical trials. These will probably be ready for production long before the stem cell lines are ever ready. Therefore, the financial motive is probably factor. Geron CEO said as much in his statement: “CEO Dr. John Scarlett told investors and analysts Tuesday that focusing on cancer drugs will allow Geron to make more money in a shorter period of time, particularly when “big pharma and big biotech companies are increasingly hungry for first-in-class cancer programs.”

With respect to the poor clinical trials, Geron press releases have revealed no such thing to date. I get Geron’s press releases, and while these are written from a source within the company that has a vested interest in making the news sound good, the phase I trials, for all intents and purposes, seemed to be going well. GRNOPC1 cells were well tolerated, and the Phase I test was meant to only test safety and not efficacy. If there were no improvements in the human patients, it seems premature to suspect that the company bailed on the basis on this one test with four patients. To suspect that the human subject experienced terrible side effects is to accuse Geron of lying, which is improper without better evidence.

Nevertheless, this is a significant story, in that ESCs do not seem to be able to compete with their adult and fetal counterparts at this time. The continued successes of cord blood, bone marrow, and other stem cell treatments are making it harder and harder for ESCs to find a niche in the market. Even in the case of spinal cord injuries, adult stem cells have made progress in helping quadriplegic patients walk with braces. Such news does not make it into the papers as often as it should, but people need to know that killing young human beings is not the most morally acceptable way to make the regenerative cures that we want.

Another factor in Geron’s decision has to be the recent decision of the European Union’s highest court that ESCs and products derived from ESCs are not patentable under applicable EU law. Because Geron probably viewed Europe as potential market for their products, this would have been a huge blow to their future marketing plans. Geron will continue to make innovative medicines, but ESC-derived products, for now, will not be one of them.