Regenerative Medicine

On February 15, 2009, in Regenerative Medicine

The genomic advances we wrote about in the last three issues are important contributors to another postmodern form of medicine: Regenerative medicine, the repair or replacement of tissue, organs, and limbs through cellular engineering inside the body or in the lab. In this issue, we digest reports of advances in regenerative medicine from just the past six months.

It may strike you, as it struck us, that many of the advances are five years from reaching the bedside. That may be true for the U.S. and other countries with strict regulations, but it is already a fact that China, Russia, and other countries are moving forward with regenerative medicine therapies in patients. We do not endorse that practice, of course, but it may have an impact on progress, one way or the other.

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It’s hard to kill an axolotl salamander. Hack off a leg, freeze a piece of its heart, even remove half of its brain, and it will simply regrow the missing or damaged organ or limb. How does it do it? It has been known for some time that cells near a wound de-differentiate from their adult state as muscle or bone cells and revert to a stem-cell-like state. The stem cells then replicate and re-differentiate into the appropriate organ or limb cells.

But to uncover this mechanism in detail requires sequencing the salamander’s genome. At about 30 billion bases, it is ten times bigger than the human genome (and we thought we were so smart!) The sequence so far has revealed that at least 10,000 genes are transcribed during regeneration, and that some 9,000 of them have related human versions. However, several thousand more do not appear to resemble any known human genes and may have evolved uniquely in salamanders. If that is so, then it could prove difficult if not impossible to program regenerative capability into humans through genetic engineering.

But we may not need to. In April 2008 the severed fingertip of a 65-year-old man was fully regenerated—nerves and all—after a month’s application of powder made from the extracellular matrix of a pig’s bladder. The matrix was obtained by removing the cells from the bladder lining, leaving the scaffold or “matrix,” which was then dried and crushed into powder.

It appears that the matrix material stimulates cells to grow, as happens with salamanders, rather than to form a protective scar—a dead end.

Clinical trials of matrix material in sheet form, for repair of resectioned tissue following oesophageal cancer surgery and for skin regrowth in burn victims, are anticipated.

Stem cells themselves appear to have anti-inflammatory properties, reducing swelling and scarring when administered to injured tissue. Bone-marrow-derived human stem cells injected into the brains of stroke-damaged mice have been shown to cause immune cells to produce chemicals that protect nerve cells, thereby reducing swelling and scarring. The search is on to uncover the mechanism involved. Reducing excessive early inflammation will be especially beneficial in treating diabetes, Alzheimer’s, and Parkinson’s disease patients.

Mice treated with stem cells experienced 60 percent less cell death compared with mice who did not receive the treatment. Furthermore, when placed in an open environment, the treated mice behaved much like healthy mice, actively exploring the space around them, unlike their more lethargic untreated counterparts.

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If slapping pig’s bladder matrix onto a wound does not appeal, one might have an alternative in a polymeric scaffold material. When seeded with heart muscle cells, the construct closely mimics the properties of heart muscle. It could be used to patch a broken heart, or in the lab to test heart drug candidates. It is flexible enough to change shape as the heart contracts and strong enough to withstand the intense contractions.

Synthetic patches that do not contain heart cells are already used to replace heart tissue surgically removed after a heart attack, and to repair congenital heart defects in infants and children. But they can lead to scarring, which is one reason why a biodegradable biomaterial with beating heart cells (in other words, an engineered tissue, such as the new material) would be a major advance.

The major hurdle still to be crossed is finding a way to mass-produce heart muscle cells in sufficient volume for the engineered tissue. Adult stem cells from bone marrow and other tissues can be turned into heart muscle cells, but not in sufficient volume using current best methods.

One solution to the volume problem might be embryonic stem cells (ESCs), which have been turned into three types of human heart cells—cardiomyocytes, endothelial cells and vascular smooth muscle cells—for transplant into mice with simulated heart disease. The transplants significantly improved heart function in the mice. The development is significant for both basic and clinical research, which can now be assured of an unlimited supply of cells to study how they develop, how they function, and how they respond to different drugs.

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Both embryonic and adult stem cell therapies are starting to show signs of success. In November, German doctors reported that they had cured a 42-year-old American man of AIDS by transplanting blood stem cells from a person naturally resistant to the AIDS virus. One expert noted, however, that the therapy would be far too expensive and remote for AIDS sufferers in the developing world and impractical even for well insured patients in the developed world.

The therapy also entails the destruction of the patient’s immune system, including bone marrow, with radiation and drugs. Ten to 30 percent of patients receiving such therapy die, but in this case, the patient also had leukemia, which justified the high risk.

The risk also turned out to be justified for five male and four female Australian patients with serious leg fractures. They re-grew thigh and shin bones within an average of four months, with very little pain, following stem cell therapy. The cells were taken from the patients’ bone marrow and cultured until there was enough volume—about 15 billion cells—to administer to the fracture sites, where the stem cells began to form bone. One patient was walking the day after the procedure, is now fully recovered, pain-free, and regularly runs and plays football.

None of the nine patients needed a second operation to get bone from somewhere else in the body. Similar trials have been conducted in the UK. Australian biotechnology company Mesoblast Limited has the worldwide license to commercialize the therapy, which is said to be between three and five years away from being used in hospitals.

With the addition of a way to re-grow cartilage, the treatment could eliminate current joint replacement surgeries.

Trauma or inflammation can cause destruction of It could also be used to repair periodontal ligament destroyed through trauma or inflammation. The ligament connects the root of a tooth to the surrounding jaw bone. Periodontal ligament cells have been implanted in a collagen network suspended between an artificial root and artificial bone, and the model was then subjected to a process to mimic the stresses of chewing. The next step may be to test the ligament in animals.

Another way to re-grow cartilage without stem cells of any sort is to grow it under high pressure. Bovine cartilage cells subjected to pressure grew, in vitro, into new tissue that had “nearly” all of the properties of natural cartilage. The technique might also work for engineering replacement bladder, blood vessel, kidney, heart valve, bone, and other tissues. As usual, it will be several years before the process will be ready for clinical testing in humans.

In anatomically related research, adult skin cells have been turned into stem cells then into artificial bone that mimics the ability of natural bone to connect with ligaments and other tissues by changing gradually from bone to softer tissue, providing better integration with the body and allowing the bone to handle weight more successfully than artificial bone lacking this property. This technology might lead to more successful outcomes for anterior cruciate ligament surgery, which often fails at the point where ligament meets bone. The tissue has been tested in animals over a period of several weeks, and further testing is in progress.

Last year, Japanese scientists achieved a similar feat with tissue taken from a child’s discarded wisdom teeth, extracted three years earlier and preserved in a freezer. Thus, there may be yet another alternative to human embryos as a source for therapeutic stem cells, although it will be at least five years before any of this research translates into therapies. (Some US dentists are reportedly offering to store stem cells taken from wisdom teeth and baby teeth (another potential source) for therapeutic purposes in the future.)

Unfortunately, such so-called induced pluripotent stem (iPS) cells require significant manipulations that increase the risk of cancer. Spermatogonial cells extracted from human testes, on the other hand, do not. They have been turned in the lab into adult germline stem cells (GSCs) with properties similar to those of embryonic stem cells, including the ability to differentiate into many different cell types. A simple testicular biopsy could thus provide the starting material for personalized regenerative medicine: stem cells that do not require embryos, are not likely to be rejected because they are made from the patient’s own cells, and can differentiate into many types of cell. GSCs from a closely related male donor could be used to treat women, but they would have to take anti-rejection medicine. Equivalent stem cells might be derived from the female reproductive tract: stem cells found on the surface of the ovary have been differentiated to become neuronal cells.

The latest method of producing stable GSC lines takes several months. Efforts are under way to speed up the process so that volumes sufficient for therapy can be produced.

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Meantime, human embryonic stem cells themselves have lost none of their promise, having been successfully turned in large quantities into red blood cells capable of carrying as much oxygen as normal blood. The ability to make blood in the lab would guarantee an ample supply of all blood types and eliminate risk from diseased donor blood in transfusions. The manufactured cells behaved like natural red blood cells in lab tests, but there remains much testing to do before the blood could be used in humans.

One day that blood could flow through artificial veins and arteries. Coronary bypass grafts have been developed by culturing human vascular smooth muscle cells and epithelial cells derived from umbilical cords in an elastic polyurethane scaffold. The significance of this particular research was not just that living tissue was successfully grafted to nonliving materials—a major breakthrough in itself—but also that the end result was stronger than in nature. One researcher said: “It is only a matter of time before human tissues can be engineered to be at least as good as the originals, and this study moves us toward that reality.”

Spinal cord tissues are likely to be among them. Although transplanting undifferentiated stem cells to repair damaged spinal cords in rats caused neuropathic pain and did not repair the damage or restore function, by first pre-differentiating the stem cells into a specific sub-type of central nervous system cells called astrocytes, transplanted animals had very high levels of new cell growth and survival, as well as recovery of limb function, without the pain.

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Still with the central nervous system: After numerous failed attempts, a congenital brain disorder has been treated successfully by a single transplantation of human neural stem cells. A more robust method for the acquisition and purification of human fetal glial progenitor cells and a new cell delivery strategy involving multiple injection sites resulted in widespread and dense donor cell engraftment throughout the central nervous system of recipient mice. The engrafted mice not only exhibited “robust, efficient and functional” myelination, but also progressive neurological improvement.

Four of the 26 experimental mice were completely rescued by the procedure; however, most of the others died within two weeks of the untreated control mice. Much work therefore lies ahead before the therapy can be tried in children, but ultimately it could lead to treatments for several hereditary and perinatal neurological disorders, including Tay-Sachs, Krabbe’s, Canavan’s, Pelizaeus-Merzbacher, Vanishing White Matter Disease, and a host of other diseases which, though individually rare, collectively kill thousands of children every year. (Shortly before this success was announced, Lorenzo Odone, whose battle with one such disease, adrenoleukodystrophy, was featured in the film Lorenzo’s Oil, passed away.) Currently there is no treatment for any of these conditions.

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In the peripheral nervous system, injured retinal cells in mice have been coaxed to create new neurons. This success could lead in the long term to treatments for macular degeneration, retinitis pigmentosa, and other diseases in which cells are lost. The therapy was a cocktail of growth factors injected into the destroyed inner retinal cells of mice, which led cells called muller glia to return to an undifferentiated stem-cell-like state. These cells then started to proliferate and a few of them differentiated into mature amacrine cells, which mediate electrical signals coming from the retinal photoreceptors and are particularly important to motion detection and night vision.

In further good news for vision restoration, last September the US Food and Drug Administration fast-tracked a treatment device for age-related macular degeneration and retinitis pigmentosa, which were hitherto essentially untreatable. The device is a capsule containing genetically engineered cells that produce a protein that may prevent light-sensitive cells in the retina from dying–thereby protecting vision. After implantation in the eye, the protein diffuses through the capsule wall and into the retina. In theory, the cells should continue to produce the protein as long as they remain alive.

In animal studies, the device slowed the degeneration of retinal cells in diseases analogous to retinitis pigmentosa and there was evidence that it could even promote retinal regeneration. A phase I (safety) human clinical trial with 10 patients revealed no safety issues. Phase II (efficacy) trials are due to conclude early in 2009.

Other approaches to these diseases include transplanting (in blind mice) embryonic stem cells directly into the retina, where it is hoped they will turn into light-sensitive cells or into pigment epithelial cells that appear to protect the light-sensitive cells; and introducing protein-producing genes into the retina.

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A milestone in regenerative medicine was passed in the Fall of 2008 with the creation of stem cells that can be developed into cell types damaged by disease, such as the insulin-producing cells in diabetes or neurons in Parkinson’s, are poised to give scientists an unprecedented view of disease.

Created through a method that does not involve human eggs or embryos, the so-called induced pluripotent stem cells (iPS) from healthy and from diseased people can be compared as they grow in the Petri dish, to find out what went wrong with the diseased stem cell. One researcher said this method would “change the way degenerative diseases are studied–we’ll reduce the whole process of disease to a petri dish.” The method can also be used to test drugs, though that might take a decade.

Unfortunately, iPS cells are made with a retrovirus that inserts transcription factors in the DNA of the patient’s skin cells. The problem is that the virus stays there and replicates itself. This can lead to cancer. New research in mice shows that an adenovirus, instead, can insert the transcription factors and then die, potentially eliminating that problem.

This could be regenerative medicine’s ethically unquestionable “magic bulletif iPS cells turn out to be as potent as human embryonic stem cells (hESCs) and if they can be derived from a patient’s own cells in sufficient numbers. The retroviral technique turns one in 1,000 cells into an iPS cell, whereas the adenovirus turns only one in every 10,000 to 100,000.

Research is under way to increase the efficiency of the adenovirus technique and to compare human iPS cells with hESCs for research and therapy. At least until that comparison is made, hESCs continue to hold the most promise.

Also last Fall, researchers found three molecular switches enabling them to reprogram a common cell found in the pancreas into the insulin-producing cells that are destroyed in childhood diabetes. The technique, developed in mice, could one day be used to grow replacement tissues not only for diabetic patients but also for those with heart disease, stroke, and other conditions.

It differs from the iPS and other stem cell approaches by converting one type of adult cell directly into another type without first transforming them into undifferentiated (i.e., stem) cells; but like them, it too is likely to take years of further development and testing before it reaches patients. The researchers hope to start human clinical trials within five years.

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Finally: A method of reliably identifying stem cells, including cancer stem cells, would be invaluable in developing a plentiful supply of cells for research into tissue regeneration, in the identification of drug targets, and in helping more accurately, reliably, and quickly to diagnose cancers. Genentech researchers have crafted such a method for prostate stem cells by first engineering whole new mouse prostates. They grafted individual prostate stem cell candidates onto the kidneys of living mice, along with some connective cells from the urogenital cavities. The result: Whole new prostates, if the candidates were genuine prostate stem cells.

Engineering new prostates was not the main goal—a new prostate would not cure the urinary incontinence and impotence that result from nerve disruption following prostate removal surgery.

In a quicker and more standard approach, a protein marker has been found that identifies adult liver stem cells, whose ability to regenerate injured liver tissue offers potential for cell-replacement therapy. Researchers have demonstrated that cells expressing the marker can differentiate into both liver cells and cells that line the bile duct. The next step will be to isolate and culture these stem cells for implant in patients whose livers can no longer repair their own tissue and whose only other option is a liver transplant.


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