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.
Scientists who work in the pharmaceutical industry have seen this time and time again: A candidate drug that works brilliantly in laboratory animals fails to work in human trials. So what’s up with this?
Now a research consortium from the University of Bonn and the biomedical company Life & Brain GmbH has shown that animal models of Alzheimer’s disease fail to recapitulate the results observed with cultured human nerve cells made from stem cells. Thus, they conclude that candidate Alzheimer’s disease drugs should be tested in human nerve cells rather than laboratory animals.
In the brains of patients with Alzheimer’s disease beta-amyloid protein deposits form that are deleterious to nerve cells. Scientists who work for drug companies are trying to find compounds that prevent the formation of these deposits. In laboratory mice that have a form of Alzheimer’s disease, over-the-counter drugs called NSAIDs (non-steroidal anti-inflammatory drugs), which include such population agents as aspirin, Tylenol, Advil, Nuprin and so on prevent the formation of beta-amyloid deposits. However in clinical trials, the NSAIDs royally flopped (see Jaturapatporn D, Isaac MG, McCleery J, Tabet N. Cochrane Database Syst Rev. 2012 Feb 15;2:CD006378).
Professor Oliver Brüstle, the director of the Institute for Reconstructive Neurobiology at the University of Bonn and Chief Executive Officer of Life and Brain GmbH, said, “The reasons for these negative results have remained unclear for a long time.”
Jerome Mertens, a former member of Professor Brüstle’s research, and the lead author on this work, said, “Remarkably, these compounds were never tested directly on the actual target cells – the human neuron.”
The reason for this disparity is not difficult to understand because purified human neurons were very difficult to acquire. However, advances in stem cell biology have largely solved this problem, since patient-specific induced pluripotent stem cells can be grow in large numbers and differentiated into neurons in large numbers.
Using this technology, Brüstle and his collaborators from the University of Leuven in Belgium have made nerve cells from human patients. These cells were then used to test the ability of NSAIDs to prevent the formation of beta-amyloid deposits.
According to Philipp Koch, who led this study, “To predict the efficacy of Alzheimer drugs, such tests have to be performed directly on the affected human nerve cells.”
Nerve cells made from human induced pluripotent stem cells were completely resistant to NSAIDs. These drugs showed no ability to alter the biochemical mechanisms in these cells that eventually lead to the production of beta-amyloid.
Why then did they work in laboratory animals? Koch and his colleagues think that biochemical differences between laboratory mice and human cells allow the drugs to work in one but not in the other. In Koch’s words, “The results are simply not transferable.”
In the future, scientists hope to screen potential Alzheimer’s disease drugs with human cells made from the patient’s own cells.
“The development of a single drug takes an average of ten years,” said Brüstle. “By using patient-specific nerve cells as a test system, investments by pharmaceutical companies and the tedious search for urgently needed Alzheimer’s medications could be greatly streamlined.”
Stem cell scientists from the University of Maryland, Baltimore have used bone marrow mesenchymal stem cells (MSCs) to treat sheep that had suffered a heart attack. They found that the injected stem cells prevented the heart from deteriorating.
This work was a collaboration between the laboratories of Mark Pittenger, ZhonGjun Wu and Bartley Griffith from the Department of Surgery and the Artificial Organ Laboratory.
After a heart attack, the region of the heart that was deprived of oxygen undergoes cell death and is replaced by a heart scar. However, the region next to the dead cells also undergo problematic changes. The cells in these regions adjacent to dead region must contract more forcibly in order to compensate for the noncontracting dead region. These cells enlarge, but some undergo cell death due to inadequate blood supply. There are other changes that can occur, such as abnormalities in Calcium ion handling and poor contractability.
Thus, the problems that result from a heart attack can spread throughout the heart and cause heart failure. In this experiment, the U of Maryland scientists injected MSCs into the sheep hearts four hours after a heart attack to determine if the stem cells could prevent the region adjacent to the dead heart cells from deteriorating.
In this experiment, bone marrow MSCs were isolated from sheep bone marrow and put through a battery of tests to ensure that they could differentiate into bone, cartilage, and fat. Once the researchers were satisfied that the MSCs were proper MSCs, they induced heart attacks in the sheep, and then injected ~200 million MSCs into the area right next to the region of the heart that died.
After 12 weeks, tissue biopsies from these sheep hearts were taken and examined. Also, the sheep hearts were measured for their heart function and structure.
The sheep that did not receive any MSC injections continued to deteriorate and showed signs of stress. The cells adjacent to the dead region expressed a cadre of genes associated with increased cell stress. Furthermore, there was increased cell death and evidence of scarring in the region adjacent to the death region. There was also evidence of Calcium ion-handling problems in the adjacent tissue and increased cell death.
On the other hand, the hearts of the sheep that had received injections of MSCs into the area adjacent to the dead region showed a reduced expression of those genes associated with increased cell stress. Also, these hearts contracted better than those that had not received stem cell injections. There was also less cell death, less scarring, and no evidence of Calcium ion-handling problems.
Changes that occur in the heart after a heart attack are collectively referred to as “remodeling.” Remodeling begins regionally, in those areas near the dead heart cells, but these deleterious changes spread to the rest of the heart, resulting in heart failure. The injections of MSCs into the area next to the dead region clearly prevented remodeling from occurring.
This pre-clinical study is a remarkable study for another reason: the MSCs used in this study were allogeneic. Allogeneic is a fancy way of saying that they did not come from the same animal that suffered the heart attack, but from some other healthy animal. Therefore, the delivery of a donor’s MSCs into the heart of a heart attack patient could potentially prevent heart remodeling.
The main problem with this experiment is that the MSCs were injected directly into the heart muscle. In humans, such a procedure requires special equipment and carries potential risks that include perforation of the heart wall, rupture of the heart wall, or further damaging the heart muscle. Therefore, if such a technology could be adapted to a more practical delivery system in humans, then certainly human clinical trials should be forthcoming.
See Yunshan Zhao, et al., “Mesenchymal stem cell transplantation improves regional cardiac remodeling following ovine infarction.” Stem Cells Translational Medicine 2012;1:685-95.
Brian Derby from the University of Manchester is using inkjet technology to distribute cells onto scaffolds that are shaped as a particular organ. Inkjet and laserjet technologies can build three-dimensional scaffolds that are coated with cells that will grow into the scaffold, assume its shape and degrade the scaffold, leaving only the tissue in its place.
This type of technology, which involves the simultaneous placement of biodegradable scaffold and cells in a three-dimensional structure that resembles that of an organ is called additive manufacture and it might very well be the future of replacement organ production. Additive manufacture recreates the biological structure in a three-dimensional, digital image, from which two-dimensional, digital slices are taken and fashioned one layer at a time. The summation of all the digital slices eventually produces a three-dimensional structure.
Inkjet technology dispenses the material that makes the scaffold in very small droplets that quickly solidify. The materials is loaded into an actual inkjet printer cartridge that is sprayed onto the surface. More droplets are placed on top of previous droplets in a very specific pattern and this repetitive distribution of droplets develop into a pattern that is very complex and forms a scaffold that nicely mimics the conditions inside the body. The scaffold also provides a surface the for cells to adhere, grow and thrive. The scaffold and its internal structure control the behavior and maintain the health of the cells embedded in the scaffold. This method of distributing cells onto a surface through a printer is called “bioprinting.”
In his article, published in the journal Science, Derby examines experiments in which porous structures are made by means of bioprinting. Bioprinting uses inkjet and laserjet technologies to distribute cells or molecules onto a surface in a desired pattern. In the case of porous structures, cells interweave throughout the scaffold and such cell-encrusted scaffolds can be placed in the body to encourage cell growth. Depending on the composition of the scaffold and the cells embedded in it, the scaffold can become a part of the body or the cells will dissolve it. Such a treatment can help heal patients with particular injuries such as cavity wounds.
Bioprinted cells can also be deposited onto scaffolds with various other chemicals, such as hormones, growth factors, or small molecules that influence the behavior of the cells. The inclusion of such molecules with the scaffold can coax cells to differentiate into distinct cell types, such as, for example, bone- or cartilage-producing cells.
Cells do suffer some damage during bioprinting, and the rule of thumb is the more energy is used to deposit the cells onto the scaffold, the lower the viability of the cells after bioprinting. To deposit and pattern cells in a scaffold there are three techniques that are used: inkjet printing, microextrusion, which is also known as filament plotting, and laser forward transfer. Bioprinting has probably the highest viability rates, and that has come after the techniques have been precisely worked out to ensure a minimum of damage. Microextrusion shows extremely variable rates of cell survival after the cells are deposited. Laser forward transfer suffers from the need for higher energy lasers to more precisely and efficiently deposit the cells, but this same higher energy kills off the cells.
Even though this technology has come a long way, it has a way to go before it is ready for the clinic. Scaffolds are being used in clinical trials, but scaffold synthesis suffers from inconsistency, and until a consistent high-quality is delivered, scaffold production will not be ready for commercial production.
Despite these caveats, there have been some successes. For example, D’Lima and others used an solution of chemicals in water (poly(ethylene glycol) dimethacrylate to be exact) that also contained cartilage-making cells (chondrocytes). They printed this suspension a bone defect in a cultured bone and then used a chemical not unlike what dentists use to harden tooth plastic called a photoinitiator. Such chemicals crosslink and bond together in response to particular wavelengths of light, and D’Lima used light to crosslink the chemicals to make a wet gel that contained the cells. After several days, this printed structure appeared to have integrated into the surrounding tissue. This experiment demonstrates that this technology is at least feasible. The hanging issue is the toxicity of the photoinitiator chemicals to cells (X. Cui, et al Tissue Eng. A 18, 1304 (2012). However, this has been studied, and it turns out the susceptibility to these chemicals is very cell type-specific. Thus, picking the right photoinitiator could potentially make this technique rather safe (see C. G. Williams, et al Biomaterials 26,1211 2005).
Scaffolds, however, can also be used to make external tissues, for example, skin patches. Derby is working with ear, nose, and throat surgeons at the Manchester Royal Infirmary. His goal is to use bioprinting to make patches that can be implanted into the inside of the nose or throat.
Derby explains: “It is very difficult to transplant even a small patch of tissue to repair the inside of the nose or mouth. Current practice, to transplant the patient’s skin to these areas, is regarded as unsatisfactory because they transplants do not possess mucous generating cells or salivary glands. We are working on techniques to print sheets of cells that are suitable for implantation in the mouth and nose.”
Derby hopes that someday bioprinting can be used to grow tumors in realistic cultures that will make superior models for drug testing and drug development.
Another term for a heart attack is a myocardial infarction (MI). A heart attack or an MI occurs when the blood supply to the heart that flows through coronary blood vessels is interrupted. The interruption of blood flow deprives the heart of nourishment and oxygen, and the downstream blood vessels and heart muscle die as a result. The decrease in blood vessel density after a MI can increase cell death, which increases the amount of cell death and the size of the heart scar. Therefore, growing more blood vessels in the heart after a heart attack, which is known as therapeutic angiogenesis, is a potentially strategy in treating an MI (see Ziebart T, et al., (2008) Circ Res 103: 1327–1334)..
To this end, a few clinical trials have attempted to used stem cells that can make blood vessels to reverse heart damage caused by an MI (see Ripa RS, et al. (2007) Circulation 116: I24–I30 and Schachinger V, et al. (2006) N Engl J Med 355: 1210–21).
Among those therapeutic agents for heart attack patients, mesenchymal stem cells (MSCs) are considered excellent candidates. MSCs have the ability to differentiate into smooth muscle, or blood vessels, which means that they can help revascularize the heart after a MI. The problem with MSCs is their tendency to die off rapidly after transplantation into the heart after a heart attack (see Ziegelhoeffer T, et al. (2004) Circ Res 94: 230–38 & O’Neill TT, et al., Circ Res 97: 1027–35; & Perry TE, et al. (2009) Cardiovasc Res 84: 317–25).
To fix this problem, MSCs can be either preconditioned before implantation (see previous posts) or genetically engineered to withstand the hostile conditions inside the heart after a heart attack.
Previously, Muhammad Ashraf and Yigang Wang from the University of Cincinnati genetically engineered MSCs to express a surface protein called CXCR4. CXC4R is the receptor for a chemokine known as CXCL12/SDF-1. SDF-1 is a rather potent stem cell recruitment molecule.
When transplanted into the hearts of rodents that had just experienced a heart attack, MSCs that expressed CXCR4 showed increased mobilization and engraftment into the damaged areas of the heart. Also, the pumping abilities of the heart regions into which the MSC-CXCR4s were infused increased, and the MSC-CXCR4 cells cranked up their secretion of blood vessel-inducing growth factors (vascular endothelial growth factor-A or VEGF-A), This led to increased formation of new blood vessels and a decrease in the early signs of left ventricular remodeling (see Zhang D, et al. (2010) Am J Physiol Heart Circ Physiol 299: H1339– H1347; Huang W, et al. (2010) J Mol Cell Cardiol 48: 702–712; &.Zhang D, et al. (2008) J Mol Cell Cardiol 44: 281–292). While these papers show truly stunning results, it was still, even after all this work, unclear if the MSCs were actually differentiating into blood vessel cells and making blood vessels.
To nail this down, Wang and his group used a clever little technique. They engineered MSCs to express CXCR4 and the viral TK gene. TK stands for “thymidine kinase,” which is an enzyme involved in nucleotide synthesis from a virus. The TK enzyme is not found in human cells, and is therefore a target for antiviral drugs. If treated with antiviral drugs that target the TK enzyme, only cells with the TK gene will be killed.
When Wang and his group used their CXCR4-engineered MSCs to treat the heart of mice that had recently suffered a heart attack, they found that their hearts improved and that these same heart were covered with new blood vessels. However, when this experiment was repeated with CXCR4-MSCs that also had the TK gene, Wang his co-workers fed the mice a drug called ganciclovir, which kills only those cells that possess the TK gene. In these mice, their heart failed to improve and also were completely devoid of the new blood vessels.
This paper nicely shows that without viable MSCs, no new blood vessels were made. This strongly suggests that the engineered MSCs are differentiating into blood vessel cells and making new blood vessels, which helps the heart recover from the heart attack and shrinks the size of the dead area of the heart.
What are the implications for human clinical trial\? This is difficult to say. Before clinical trials with genetically engineered cells are approved those cells will need to go through piles of safety tests before they can be used in clinical trials. Once that hurdle is passed, then they can be used in human clinical trials, and they will certainly prove efficacious for human patients.
Tissue engineers have designed scaffolds for stem cells made from nanotubes that induce them to form cartilage. These nanofibers are tiny, artificial fiber scaffolds that are thousands of times smaller than a human hair. Cartilage formation has succeeded in laboratory and animal model systems.
Much more work is necessary before these scaffolds can be used inside a human body; the results of this present study hold promise for designing new techniques to help millions who endure joint pain.
Jennifer Elisseeff, Ph.D., Jules Stein Professor of Ophthalmology and director of the Translational Tissue Engineering Center at the Johns Hopkins University School of Medicine, said: “Joint pain affects the quality of life of millions of people. Rather than just patching the problem with short-term fixes, like surgical procedures such as microfracture, we’re building a temporary template that mimics the cartilage cell’s natural environment, and taking advantage of nature’s signals to biologically repair cartilage damage,”
Unfortunately, cartilage does not repair itself when damaged. For the last decade, Elisseeff’s research team has been trying to better understand the development and growth of cartilage cells (chondrocytes). Part of these studies has involved building scaffolds that mimics the environment inside the body that produces new cartilage tissue. The cartilage-making environment consists of a three-dimensional mix of protein fibers and gel. This matrix provides support to connective tissue throughout the body, and physical and biological cues for cells to grow and differentiate.
In the laboratory, the Elisseeff team created a nanofiber-based network that utilized a process called electrospinning. Electrospinning shoots a polymer stream onto a charged platform, to which a compound called chondroitin sulfate is added. Chondroitin sulfate is commonly found in many joint supplements. After characterizing the manufactured fibers, they made several different scaffolds from spun polymer or spun polymer plus chondroitin sulfate. Elisseeff and her colleagues then seeded the scaffolds with bone marrow stem cells from goats in order to test how well the scaffolds supported the growth of the stem cells.
The results showed that the scaffold-supported stem cells formed more voluminous, cartilage-like tissues that those grown without the manufactured scaffolds.
“The nanofibers provided a platform where a larger volume of tissue could be produced,” said Elisseeff, adding that 3-dimensional nanofiber scaffolds were more useful than the more common nanofiber sheets for studying cartilage defects in humans.
Next, Elisseeff and her group tested the ability of these nanotube scaffolds and the cartilage they produce in an animal model. They implanted the nanofiber scaffolds into damaged cartilage in the knees of rats. They compared the quality of the knee repair to damaged cartilage in knees that were not treated with any stem cells. The nanofiber scaffolds improved tissue development and repair. The nanofiber-implanted knees produced far more cartilage as measured by the production of collagen, which is a component of cartilage. The nanofiber scaffolds induced the production of larger quantities of a more durable type of collagen, which is typically absent in surgically repaired cartilage tissue. In rats, the limbs with damaged cartilage treated with nanofiber scaffolds generated a higher percentage of the more durable collagen (type 2) than those damaged areas that were left untreated.
“Whereas scaffolds are generally pretty good at regenerating cartilage protein components in cartilage repair, there is often a lot of scar tissue-related type 1 collagen produced, which isn’t as strong,” said Elisseeff. “We found that our system generated more type 2 collagen, which ensures that cartilage lasts longer. Creating a nanofiber network that enables us to more equally distribute cells and more closely mirror the actual cartilage extracellular environment are important advances in our work and in the field. These results are very promising.”